Use of reagent for inhibiting nasopharyngeal carcinoma stemness molecular marker in preparation of nasopharyngeal carcinoma adjuvant therapy drug

Abstract

The application discloses application of a reagent for inhibiting nasopharyngeal carcinoma stemness molecular marker in preparation of a nasopharyngeal carcinoma adjuvant treatment drug. The treatment is preferably nasopharyngeal carcinoma targeted treatment or stemness treatment. The application identifies a new type of molecular marker capable of reflecting nasopharyngeal carcinoma drug treatment sensitivity and finds a drug capable of effectively inhibiting nasopharyngeal carcinoma progression from tumor stemness. Specifically, PSMC3IP, NABP2, CDC45 or HJURP expression can reflect the stemness state of nasopharyngeal carcinoma, the higher the PSMC3IP, NABP2, CDC45 or HJURP expression, the higher the stemness degree of the nasopharyngeal carcinoma patient, the poorer the prognosis, and the poorer the effect of receiving traditional chemotherapy and radiotherapy, and the easier the resistance to the chemotherapy drug. By using siRNA technology to inhibit the expression of PSMC3IP, NABP2, CDC45 or HJURP, the tumor cell proliferation ability can be reduced, and the tumor stemness of nasopharyngeal carcinoma is reduced, so that the purpose of treating nasopharyngeal carcinoma is achieved. The application provides a new action target and effective drug for the treatment of nasopharyngeal carcinoma.

Description

Technical Field

[0001] This invention relates to the application of a reagent for inhibiting stem molecular markers of nasopharyngeal carcinoma in the preparation of adjuvant drugs for nasopharyngeal carcinoma. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a type of head and neck tumor with a unique geographical distribution, prevalent in southern my country, particularly Hainan and Guangdong provinces. Although chemotherapy and chemotherapy-based treatments can effectively control the development of most NPC cases, about 10% of NPC patients experience local or regional lymph node recurrence after treatment. Patients with recurrence have a poorer survival prognosis and often develop resistance to chemotherapy drugs. Cancer stem cells are a type of cancer cell characterized by self-renewal, unlimited proliferation, multi-lineage differentiation, and tumor initiation. Due to their extensive proliferative capacity, these cells can promote the development of diffuse metastatic tumors and are a major cause of tumor progression, metastasis, and treatment resistance.

[0003] Therefore, starting from tumor stemness, identifying novel molecular markers that can reflect the sensitivity of nasopharyngeal carcinoma to drug treatment and finding drugs that can effectively inhibit the progression of nasopharyngeal carcinoma are currently the focus of research. Summary of the Invention

[0004] One objective of this invention is to provide a drug that can inhibit the progression of nasopharyngeal carcinoma;

[0005] The second objective of this invention is to provide the application of this drug in the adjuvant treatment of nasopharyngeal carcinoma.

[0006] To achieve the above objectives, the present invention employs the following technical means:

[0007] The inventors of this invention performed consistent clustering of nasopharyngeal carcinoma (NPC) patients based on activity scores from multiple stem cell gene datasets to determine the stem subtype of NPC tumors and identify key molecular markers within this subtype. The study found that patients with higher stemness scores had poorer prognoses and were more likely to be in advanced stages. PSMC3IP (PSMC3 Interacting Protein), NABP2 (Nucleic Acid Binding Protein 2), CDC45 (Cell Division Cycle 45), and HJURP (Holliday Junction Recognition Protein) were elevated in the stem subtype, and their high expression is a risk factor for NPC prognosis. Therefore, PSMC3IP, NABP2, CDC45, and HJURP are identified as novel molecular markers for NPC stemness. Furthermore, siRNA technology was used to successfully interfere with the expression of PSMC3IP, NABP2, CDC45, and HJURP in nasopharyngeal carcinoma carcinoma lines. The effects of these genes on nasopharyngeal carcinoma stem cell activity were observed using a tumor stem cell spheroidization assay. The spheroidization efficiency results showed that tumor cell stemness was weakened after interference with PSMC3IP, NABP2, CDC45, and HJURP. In addition, by detecting the protein expression levels of known tumor stemness markers (LGR5, CD44, SOX2, NANOG, and OCT4), it was found that knocking out PSMC3IP, NABP2, CDC45, and HJURP in nasopharyngeal carcinoma carcinoma lines downregulated the expression of most tumor stemness markers. Only interference with CDC45 did not change CD44 expression, and interference with NABP2 did not change SOX2 expression. CCK8 assay results showed that knocking out PSMC3IP, NABP2, CDC45, or HJURP reduced tumor cell proliferation. The above results indicate that inhibiting the expression of PSMC3IP, NABP2, CDC45 and HJURP can suppress the tumor stemness of nasopharyngeal carcinoma, thereby achieving the goal of treating nasopharyngeal carcinoma.

[0008] Based on the above research, this invention proposes the application of reagents for inhibiting nasopharyngeal carcinoma stem molecular markers in the preparation of adjuvant drugs for nasopharyngeal carcinoma, wherein the nasopharyngeal carcinoma stem molecular markers are at least one of PSMC3IP, NABP2, CDC45 and HJURP.

[0009] Preferably, the treatment is targeted therapy or dry therapy for nasopharyngeal carcinoma.

[0010] Preferably, the reagent used to inhibit the nasopharyngeal carcinoma stem molecular marker is an siRNA targeting at least one of PSMC3IP, NABP2, CDC45, and HJURP.

[0011] Preferably, the siRNA sequences for inhibiting PSMC3IP are as shown in SEQ ID NO. 11 and 12, SEQ ID NO. 13 and 14, or SEQ ID NO. 15 and 16; the siRNA sequences for inhibiting NABP2 are as shown in SEQ ID NO. 17 and 18, SEQ ID NO. 19 and 20, or SEQ ID NO. 21 and 22; the siRNA sequences for inhibiting CDC45 are as shown in SEQ ID NO. 23 and 24, SEQ ID NO. 25 and 26, or SEQ ID NO. 27 and 28; and the siRNA sequences for inhibiting HJURP are as shown in SEQ ID NO. 29 and 30, SEQ ID NO. 31 and 32, or SEQ ID NO. 33 and 34.

[0012] Compared with the prior art, the beneficial effects of the present invention are:

[0013] This invention focuses on tumor stemness, identifying novel molecular markers that reflect the sensitivity of nasopharyngeal carcinoma (NPC) to drug therapy and identifying drugs that can effectively inhibit NPC progression. Specifically, the expression of PSMC3IP, NABP2, CDC45, or HJURP reflects the stemness status of NPC. Higher expression levels of these markers indicate a higher degree of stemness in NPC patients, a worse prognosis, poorer response to traditional chemotherapy and radiotherapy, and a greater likelihood of developing resistance to chemotherapy drugs. By using siRNA technology to inhibit the expression of PSMC3IP, NABP2, CDC45, or HJURP, the proliferative capacity of tumor cells can be reduced, thus decreasing the tumor stemness of NPC and achieving the goal of treating NPC. This invention provides new therapeutic targets and effective drugs for NPC treatment. Attached Figure Description

[0014] Figure 1 For the identification of the dry subtype of nasopharyngeal carcinoma.

[0015] The following are included: A. Consistent cluster analysis of nasopharyngeal carcinoma. B. Distribution of StemChecker stemness scores among nasopharyngeal carcinoma subtypes. C. Distribution of mRNAsi stemness scores among nasopharyngeal carcinoma subtypes. D. Prognostic analysis of glioma subtypes.

[0016] Figure 2 For the identification of the nasopharyngeal carcinoma stem module.

[0017] Among them, A. A clustering dendrogram was constructed using weighted correlation coefficients to cluster genes with similar expression patterns into co-expression modules, with each color representing a module. B. The association between nasopharyngeal carcinoma gene modules and clinical phenotypes. C. The association between network module genes and the C2 subtype.

[0018] Figure 3 The impact of PSMC3IP on the development and progression of nasopharyngeal carcinoma.

[0019] Among them, A. the effect of PSMC3IP expression level on progression-free survival in nasopharyngeal carcinoma. B. the effect of PSMC3IP expression on disease-free survival in head and neck squamous cell carcinoma. C. PSMC3IP is significantly highly expressed in the C2 stem subtype of nasopharyngeal carcinoma. D. PSMC3IP is significantly highly expressed in head and neck squamous cell carcinoma tumor samples.

[0020] Figure 4 The impact of NABP2 on the development and progression of nasopharyngeal carcinoma.

[0021] Among them, A. the effect of NABP2 expression level on progression-free survival in nasopharyngeal carcinoma. B. the effect of NABP2 expression on disease-free survival in head and neck squamous cell carcinoma. C. NABP2 is significantly highly expressed in the C2 stem subtype of nasopharyngeal carcinoma. D. NABP2 is significantly highly expressed in head and neck squamous cell carcinoma tumor samples.

[0022] Figure 5 The impact of CDC45 on the development and progression of nasopharyngeal carcinoma.

[0023] Among them, A. The effect of CDC45 expression level on progression-free survival in nasopharyngeal carcinoma. B. The effect of CDC45 expression on disease-free survival in head and neck squamous cell carcinoma. C. CDC45 is significantly highly expressed in the C2 stem subtype of nasopharyngeal carcinoma. D. CDC45 is significantly highly expressed in head and neck squamous cell carcinoma tumor samples.

[0024] Figure 6 The impact of HJURP on the occurrence and development of nasopharyngeal carcinoma.

[0025] Among them, A. the effect of HJURP expression level on progression-free survival in nasopharyngeal carcinoma; B. the effect of HJURP expression on disease-free survival in head and neck squamous cell carcinoma; C. significantly high expression of HJURP in the C2 stem subtype of nasopharyngeal carcinoma; and D. significantly high expression of HJURP in head and neck squamous cell carcinoma tumor samples.

[0026] Figure 7 This study confirms the tumor stemness of PSMC3IP, NABP2, CDC45, and HJURP in nasopharyngeal carcinoma cell lines.

[0027] Among them, A. RT-qPCR experiments demonstrated increased RNA expression levels of PSMC3IP, NABP2, CDC45, and HJURP in nasopharyngeal carcinoma cell lines. B. Western blotting experiments demonstrated increased protein expression levels of PSMC3IP, NABP2, CDC45, and HJURP in nasopharyngeal carcinoma cell lines. C. Expression levels of PSMC3IP, NABP2, CDC45, and HJURP in nasopharyngeal carcinoma cell lines after siRNA interference. D. Images of HNE1 and CNE2 tumor stem cell spheroidization experiments after NC and siRNA transfection; bar graphs show the spheroidization rate of nasopharyngeal carcinoma cells. E. Western blotting analysis showed decreased protein expression of tumor stemness markers LGR5, CD44, SOX2, NANOG, and OCT4 in nasopharyngeal carcinoma cell lines after siRNA transfection. F. CCK-8 assay confirmed decreased tumor cell proliferation after siRNA transfection. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Determination of novel dry molecular markers for nasopharyngeal carcinoma

[0030] 1. Identification of nasopharyngeal carcinoma subtypes based on stem cell gene sets

[0031] The nasopharyngeal carcinoma transcriptome sequencing data used in the experiment were collected from the Gene Expression Omnibus (GEO) database GSE102349, and the head and neck squamous cell carcinoma dataset used for the validation set was collected from the Cancer Genome Atlas (TCGA, https: / / portal.gdc.cancer.gov / ) database. First, stem cell-related gene sets were collected from the Molecular Signatures Database (MSigDB), and nasopharyngeal carcinoma patients were scored using the single-sample gene enrichment analysis method (ssGSEA). ConsensusClusterPlus R package was used to perform consensus clustering based on the score level of the stem cell gene set in the samples, thereby obtaining the nasopharyngeal carcinoma stem subtypes C1 and C2. Figure 1A). Stemility was assessed in the C1 and C2 subtypes using an external stemness-related gene set provided in StemChecker and stemness indices (mRNAsi) based on logistic regression machine learning. The rank-sum test showed that multiple stemness indices for the C2 subtype were significantly higher than those for the C1 subtype, indicating that the C2 subtype tends to exhibit characteristics of tumor stem cells. Figure 1 B and Figure 1 C). Furthermore, survival analysis using the Kaplan-Meier test revealed poorer progression-free survival in the C2 subtype, indicating that nasopharyngeal carcinoma patients with higher dryness indices are more prone to recurrence. Figure 1 D).

[0032] 2. WGCNA analysis to identify the stem type of nasopharyngeal carcinoma.

[0033] Based on the top 10,000 genes by absolute median in nasopharyngeal carcinoma transcriptome sequencing data, weighted correlation network analysis (WGCNA) was used to identify key network modules. C1, C2, and mRNAsi stemness scores were combined to assess the association between network modules and clinical phenotypes. Figure 2 A). The results showed that the blue module was negatively correlated with the C1 subtype and significantly positively correlated with the C2 subtype and mRNAsi score, indicating that it is a nasopharyngeal carcinoma stem module. Figure 2 B). Genes with a correlation greater than 0.4 with the C2 phenotype and a correlation greater than 0.6 with the module were ultimately selected as key genes for nasopharyngeal carcinoma stem cell lineage, resulting in a total of 141 key genes. Figure 2 C).

[0034] 3. Confirm the effects of PSMC3IP, NABP2, CDC45, and HJURP on the stemness of nasopharyngeal carcinoma.

[0035] The impact of key genes on progression-free survival in nasopharyngeal carcinoma was assessed using univariate Cox analysis combined with the Kaplan-Meier test. The prognostic hazard ratios (HRs) and log-rank P-values ​​for 37 genes were both significant (P < 0.05). Using TCGA head and neck squamous cell carcinoma data as external validation, high expression of PSMC3IP, NABP2, CDC45, and HJURP was found to be a risk factor for both progression-free survival in nasopharyngeal carcinoma and disease-free survival in head and neck squamous cell carcinoma. Figure 3 A, 4A, 5A, 6A and Figure 3 B, 4B, 5B, 6B). Differential expression analysis showed that PSMC3IP, NABP2, CDC45, and HJURP were significantly increased in both nasopharyngeal carcinoma C2 subtype and head and neck squamous cell carcinoma. Figure 3 C, 4C, 5C, 6C and Figure 3D, 4D, 5D, 6D, rank-sum test p-value less than 0.05).

[0036] 4. Clinical auxiliary diagnosis and prognosis of nasopharyngeal carcinoma

[0037] In clinical practice, the expression levels of PSMC3IP, NABP2, CDC45 and HJURP can be detected to assess the dryness characteristics and prognosis of nasopharyngeal carcinoma patients.

[0038] The methods for detecting the expression levels of PSMC3IP, NABP2, CDC45, and HJURP are as follows:

[0039] 1. RNA extraction

[0040] Cell sample: Nasopharyngeal carcinoma cells. Add 300 μL each of lysis buffer and diluent to the nasopharyngeal carcinoma cell pellet, mix thoroughly, and let stand for 5 min. Transfer Trizol to a 1.5 mL centrifuge tube and centrifuge at 10000-12000 rpm for 5 min. Collect the supernatant, add 300 μL of anhydrous ethanol, shake well, remove the flocculent material, transfer to an adsorption column, and centrifuge at 10000-12000 rpm for 1 min. Discard the filtrate, add 600 μL of RNA wash buffer, centrifuge at 10000-12000 rpm for 45 s, discard the filtrate again, add 50 μL of incubation buffer, incubate in the center of the membrane for 15 min, add 600 μL of RNA wash buffer, centrifuge at 10000-12000 rpm for 45 s, repeat once, and centrifuge an empty tube at 10000-12000 rpm for 2 min. Add 50-70 μL of nuclease-free water, let stand for 2 min, then centrifuge at 10000-12000 rpm for 1 min to collect total RNA and store at -80℃.

[0041] 2. Reverse transcription and qRT-PCR

[0042] Reverse transcription was performed using the SuperMix for qPCR (gDNAdigesterplus) reverse transcription kit. The sample loading consisted of 10 μL RNase-free ddH2O, 2 μL 5×RT-buffer, 1 μL gDNAdigester, and 1-5 ng RNA, with the volume brought to 10 μL using RNase-free water. The reverse transcription reaction program was 25°C for 5 min; 42°C for 30 min; and 85°C for 5 min. The reverse-transcribed cDNA was stored at 4°C. qRT-PCR was performed using the Green Master Mix qPCR kit. The reaction mixture included... qPCR 10 μL Green Master Mix, 0.4 μL primers, 3.6 μL ddH2O, and 1 μL sample template were prepared. Different common reaction systems were configured according to the different genes being amplified. First, the common reaction system was added to a 96-well PCR plate, then the cDNA was added. Finally, the plate was sealed with sealing film and centrifuged at 1000 rpm for 1 min. The 96-well plate was then placed on a PCR instrument for amplification. The reaction steps were: 95℃, 5 min (pre-denaturation, cycle 1); 95℃, 10 s (denaturation); 60℃, 30 s (cycle 40). The primers used for qRT-PCR are shown in Table 1 below. These primers can be used to detect PSMC3IP, NABP2, CDC45, and HJURP transcripts.

[0043] Table 1. Primer sequences

[0044]

[0045] Results: High expression of PSMC3IP, NABP2, CDC45, or HJURP indicated that the nasopharyngeal carcinoma patient exhibited characteristics of tumor stem cells, had a poor prognosis, and was resistant to chemotherapy. Low expression of PSMC3IP, NABP2, CDC45, or HJURP indicated a better prognosis and sensitivity to chemotherapy.

[0046] Example 2: Application of reagents for inhibiting the stem molecular marker of nasopharyngeal carcinoma in targeted therapy or stem therapy for nasopharyngeal carcinoma.

[0047] Molecular biology experiments were used to verify the effects of inhibiting PSMC3IP, NABP2, CDC45, and HJURP on the stemness of nasopharyngeal carcinoma.

[0048] Based on the computational analysis results, we simultaneously validated PSMC3IP, NABP2, CDC45, and HJURP using molecular biology experiments. The RNA and protein expression levels of PSMC3IP, NABP2, CDC45, and HJURP were examined in nasopharyngeal carcinoma cell lines (HNE1, CNE2, 5-8F, and SUNE-1) and the normal nasopharyngeal epithelial cell line (N69), respectively. The specific methods are as follows:

[0049] 1. RT-qPCR experimental method

[0050] Total RNA was extracted from glioma cells using the EastepSuper Total RNA Extraction Kit (Promega LS1040). The total RNA was reverse transcribed into cDNA using the SuperMix for qPCR (gDNA digester plus) (YEASEN H7101160) reverse transcription kit. The relative expression of PSMC3IP, NABP2, CDC45, and HJURP was then detected using qPCR Green Master Mix (YEASEN H6103020) according to the manufacturer's protocol. The expression levels of the target genes were normalized using the internal control GAPDH. Data are expressed as fold changes relative to the cycle threshold (CT value).

[0051] 1) Total RNA extraction

[0052] RNA extraction was performed using the EastepSuper Total RNA Extraction Kit.

[0053] (1) Add 300 μL of lysis buffer and diluent to the glioma cell pellet, mix thoroughly, and let stand for 5 min.

[0054] (2) Centrifuge at 10000-12000r / min for 5min and collect the supernatant.

[0055] (3) Add 300 μL of anhydrous ethanol, shake well, and after aspirating the flocculent material, transfer it into the adsorption column.

[0056] (4) Centrifuge at 10000-12000r / min for 1min.

[0057] (5) Discard the filtrate and add 600 μL of RNA washing solution.

[0058] (6) Centrifuge at 10000-12000 r / min for 45 s.

[0059] (7) Discard the filtrate and add 50 μL of incubation solution to the center of the membrane and incubate for 15 min.

[0060] (8) Add 600 μL of RNA washing buffer, centrifuge at 10,000-12,000 rpm for 45 seconds, repeat once, and centrifuge an empty tube at 10,000-12,000 rpm for 2 minutes.

[0061] (9) Add 50-70 μL of nuclease-free water, let stand for 2 min, then centrifuge at 10000-12000 r / min for 1 min.

[0062] (10) Collect total RNA and store at -80℃.

[0063] 2) RNA reverse transcription

[0064] RNA reverse transcription was performed using the SuperMix for qPCR (gDNAdigesterplus) kit.

[0065] (1) Remove residual genomic DNA and incubate at 42°C for 2 min. The reagent loading system is shown in Table 2 below.

[0066] Table 2. Reverse transcription incubation solution loading system

[0067]

[0068]

[0069] (2) The reverse transcription reagent loading system is shown in Table 3 below.

[0070] Table 3 Reverse Transcription Reaction System

[0071]

[0072] (3) The temperature system is shown in Table 4 below.

[0073] Table 4 Reverse Transcription Temperature System

[0074]

[0075] (4) The reverse transcribed cDNA is stored at 4°C.

[0076] 3) Q-PCR

[0077] Q-PCR was performed using the qPCR Green Master Mix kit.

[0078] (1) The sampling system is shown in Table 5 below:

[0079] Table 5 qPCR reaction system

[0080]

[0081] (2) The temperature system settings are shown in Table 6 below:

[0082] Table 6 Temperature System

[0083]

[0084]

[0085] 2. siRNA transfection

[0086] The designed siRNA sequences targeting PSMC3IP, NABP2, CDC45, and HJURP are shown in Table 7.

[0087] Table 7. siRNA Sequences

[0088]

[0089]

[0090] method:

[0091] (1) Cell plating

[0092] (2) Replace the culture medium in the 6-well plate with a serum- and antibiotic-free medium.

[0093] (3) Complex preparation and transfection: Before use, mix the GND reagent (Gedina Biotechnology) (10 μg / μl); add 100 μl of serum-free culture medium and 4 μl of siRNA (1 μg) to a 1.5 ml sterile centrifuge tube and mix with a pipette; at the same time, add 80 μl of serum-free culture medium to another 1.5 ml sterile centrifuge tube, add 20 μl of GND reagent (10 μg / μl), mix with a pipette, add to the siRNA-culture medium mixture and mix well. After standing at room temperature for 15 min, transfect immediately.

[0094] (4) Add 200 μl of the transfection mixture to each well, bringing the final volume to 1 ml. The final concentration of GND reagent is 200 μg / ml. After adding the mixture, gently shake the plate to distribute it evenly. Incubate the cells at 37°C for 6-8 hours, then replace with complete culture medium.

[0095] 3. Western blot (WB) experimental method

[0096] 1) Rubber Mixing

[0097] (1) Washing the glass plate: Wash with dish soap, rinse with distilled water, rinse with anhydrous ethanol, let it air dry (or use a hair dryer to dry).

[0098] (2) Preparation of separating gel: Prepare a 5 ml / gel separating gel. Using a suitable pipette, pipette 1.9 ml / gel DDW, 1.7 ml / gel 30% Acr-Bis (29:1), 1.3 ml / gel separating gel buffer, 0.05 ml / gel 10% APS, and 0.002 ml / gel TEMED into an EP tube. Mix well with a shaker and place in a fume hood.

[0099] (3) Install the glue-making rack: Place the glass short plate and long plate on the glue-making rack, making sure the bottom layers of the two plates are aligned and clamped, and move them to the fume hood for glue injection.

[0100] (4) Injecting the separating gel: Use a dropper to draw up the separating gel and carefully inject it until the upper edge of the separating gel is about 2cm from the upper edge of the long plate. Inject ultrapure water into the top layer and pour out the ultrapure water after gelation.

[0101] (5) Prepare stacking gel: Prepare a 2 ml / gel separating gel. Using a suitable pipette, pipette 1.15 ml / gel DDW, 0.33 ml / gel 30% Acr-Bis (29:1), 0.5 ml / gel stacking gel buffer, 0.02 ml / gel 10% APS, and 0.002 ml / gel TEMED into an EP tube. Mix well with a shaker and place in a fume hood.

[0102] (6) Injecting the stacking gel: Use a dropper to inject the stacking gel into the reserved 2cm gap, carefully insert the sample comb, and be careful not to leave any air bubbles. Wait for the gel to form.

[0103] 2) Sample loading and electrophoresis

[0104] (1) Take the glass plate out of the gel casting frame, place it in the pre-cleaned electrophoresis tank and fix it, pull out the comb vertically, add enough electrophoresis solution to the inner tank, flush out the air bubbles in the sample loading well, and add the sample and marker to the sample loading well using the appropriate pipette according to the required sample volume.

[0105] (2) After loading the sample, add electrophoresis solution to the corresponding mark (2 plates or 4 plates) in the outer electrophoresis tank, and cover the electrophoresis tank (note the positive and negative electrodes).

[0106] (3) Turn on the power and perform electrophoresis at 80V. After the lower edge of the sample is flush, adjust the voltage to 120V and wait for about 90 minutes for the bromophenol blue to reach the position where it will exit the gel.

[0107] 3) Transfer membrane

[0108] (1) Prepare a 0.45μm or 0.22μm PVDF membrane of appropriate size (pore size is determined according to the molecular weight of the target protein; for small molecules below 20kDa, a membrane with a small pore size can be selected) and four sheets of filter paper. Soak them in the electroporation solution. Activate the PVDF membrane in methanol first.

[0109] (2) Peel off the glass plate and place the adhesive in the electrophoresis solution to wash away any residual electrophoresis solution. Then carefully place it on the electrophoresis clamp, ensuring there are no air bubbles. Place the black plate (negative electrode) of the electrophoresis clamp at the bottom and install it in the following order: "sponge-filter paper-adhesive-PVDF membrane-filter paper-sponge". Avoid air bubbles at each step.

[0110] (3) After clamping, place it in the electroporation cell (the electroporation cell is placed in a plastic basin with ice beforehand), fill it with electroporation solution and add ice, and transfer the membrane at 295mA for 1.5h or 2h (the transfer time depends on the molecular weight of the target protein; for proteins larger than 200kDa, the transfer time can be appropriately extended).

[0111] 4) Closed

[0112] Soak in 5% skim milk for 1 hour at 60 rpm.

[0113] 5) Antibody incubation

[0114] Add primary antibody: Wash the PVDF membrane three times with TBST solution at 90 r / min for 5 min. Determine the cutting position of the membrane according to the molecular weight of the target protein and the internal reference protein. Put it into the antibody incubation bag and add an appropriate amount of primary antibody solution (3 ml). Seal it carefully, do not squeeze the PVDF membrane, and put it in the refrigerator at 4°C overnight.

[0115] 6) Add secondary antibody:

[0116] After overnight incubation, remove the incubation bag and place it on a shaker to return to room temperature for 30 minutes at 60 rpm. Recover the primary antibody and remove the PVDF membrane (note that the tweezers should be held at the edge of the membrane). Wash the membrane three times with TBST solution for 5 minutes each time. Add the secondary antibody solution and incubate on a shaker for 90 minutes. After incubation, wash the membrane three times with TBST solution for 10 minutes each time.

[0117] 7) ECL development

[0118] 4.4 Ball-forming experimental method

[0119] (1) Use trypsin-EDTA solution (T3924) to digest cells with a fusion degree of 80-90% and in good condition.

[0120] (2) Resuspend the cells at 1000 rpm for 5 min and aspirate the supernatant. Resuspend the cells in culture medium.

[0121] (3) Cell counting, using ultra-low adsorption cell culture plates ( Cell culture using ultra-low adsorption multi-well plates (approximately 1000 cells per well in a 6-well plate).

[0122] (4) Culture for about 10 days, observe the cell spheroidization and calculate SPF.

[0123] (5) Secondary ball formation:

[0124] Cell spheres were collected by filtration through a 70 μm cell sieve, digested with trypsin, and digestion was terminated with serum-free medium. Cells were washed twice with PBS, resuspended, counted, and cultured in ultra-low adsorption cell culture plates (approximately 1000 cells per well in a 6-well plate) for about 10 days. Cell sphere formation was observed and SPF was calculated. SFE was calculated as follows: SFE = Number of cell spheres larger than 75 μm in diameter per well / Total number of original seeded cells per well.

[0125] 4. CCK8 Experiment

[0126] (1) Nasopharyngeal carcinoma cells were prepared into a cell suspension and counted using a cell counter. The cell count was based on a concentration of 3.5 × 10⁻⁶ cells / mL. 3 Cells / well were seeded into 96-well cell culture plates and incubated at 37°C with 5% CO2 for 24 hours.

[0127] (2) After the cells adhered, culture medium of the appropriate concentration was added. A negative control group, different concentrations, and a blank control group were set up. The 96-well plates were then placed in a 37°C, 5% CO2 cell culture incubator. After 24 hours, 10 μL of CCK8 solution was added to each well of the 96-well cell culture plate using a repeat pipette. The culture plate was incubated in the incubator for 1 hour. The absorbance at 450 nm was measured using a microplate reader.

[0128] result:

[0129] RT-qPCR results showed that PSMC3IP, NABP2, CDC45, and HJURP were significantly increased in nasopharyngeal carcinoma cell lines. Furthermore, PSMC3IP, HJURP, and CDC45 were highly expressed in the HNE1 cell line, while NABP2 was highly expressed in the CNE2 cell line. Therefore, subsequent experiments were conducted in these two cell lines. Figure 7 A, Figure 7 B). The expression of PSMC3IP, NABP2, CDC45, and HJURP was successfully interfered with in nasopharyngeal carcinoma cell lines using siRNA technology. Figure 7 C). NC and siRNA were transfected into nasopharyngeal carcinoma cells, and the effects of these genes on nasopharyngeal carcinoma stemness were observed using a tumor stem cell spheroidization assay. The results showed that interference with PSMC3IP, NABP2, CDC45, and HJURP reduced tumor cell stemness. Figure 7 D). Furthermore, by detecting the protein expression levels of known tumor stemness markers (LGR5, CD44, SOX2, NANOG, and OCT4), it was found that the expression of most tumor stemness markers was downregulated after knocking out PSMC3IP, NABP2, CDC45, and HJURP in nasopharyngeal carcinoma cell lines. Only after interfering with CDC45 did CD44 expression remain unchanged, and after interfering with NABP2 did SOX2 expression remain unchanged. Figure 7 E). CCK8 assay results showed that knockout of PSMC3IP, NABP2, CDC45, or HJURP reduced tumor cell proliferation. Figure 7 F). The above results indicate that PSMC3IP, NABP2, CDC45, and HJURP can promote the stemness of nasopharyngeal carcinoma tumors and are novel molecular markers of nasopharyngeal carcinoma stemness. At the same time, inhibiting the expression of PSMC3IP, NABP2, CDC45, or HJURP can reduce the proliferation capacity of tumor cells and decrease the stemness of nasopharyngeal carcinoma, thereby achieving the goal of treating nasopharyngeal carcinoma.

Claims

1. The application of a reagent for inhibiting nasopharyngeal carcinoma stem molecular markers in the preparation of adjuvant drugs for nasopharyngeal carcinoma, wherein the nasopharyngeal carcinoma stem molecular markers are composed of NABP2, CDC45, and HJURP, and the reagent for inhibiting the nasopharyngeal carcinoma stem molecular markers is siRNA targeting NABP2, CDC45, and HJURP; wherein, The sequences of siRNAs used to inhibit NABP2 are shown in SEQ ID NO. 33 and 34; the sequences of siRNAs used to inhibit CDC45 are shown in SEQ ID NO. 27 and 28; and the sequences of siRNAs used to inhibit HJURP are shown in SEQ ID NO. 21 and 22.

2. The application as described in claim 1, characterized in that, The treatment described is targeted therapy or dry therapy for nasopharyngeal carcinoma.

Images