Shmt1 and use thereof in radiotherapy of head and neck tumors

Abstract

The application discloses SHMT1 and application thereof in radiotherapy of head and neck tumors, relates to the field of biology, and discloses a new mechanism that low expression of SHMT1 enhances tumor stem cell characteristics by activating GLI1 and downstream stemness-related genes, and then promotes DNA damage repair and radiotherapy resistance, based on the unique regulation of SHMT1 in radioresistant hypopharyngeal squamous cell carcinoma. Experiments prove that knocking down SHMT1 can reduce the sensitivity of tumors to radiotherapy, and overexpression of SHMT1 effectively inhibits tumor growth in vitro and in vivo, indicating that SHMT1 is not only a key biomarker for judging the prognosis of HSCC radiotherapy, but also an intervention target with great transformation potential. The SHMT1 expression regulating material provided by the application can be used for preparing a drug composition for improving the radiotherapy effect and reducing the recurrence risk, or for screening candidate compounds for reversing radiotherapy resistance, and lays a foundation for developing a combined treatment strategy based on the interaction of one-carbon metabolism and tumor stemness, and is expected to improve the long-term survival rate of HSCC patients.

Description

Technical Field

[0001] This invention relates to biotechnology, specifically to SHMT1 and its application in radiotherapy for head and neck tumors. Background Technology

[0002] Hypopharyngeal squamous cell carcinoma (HSCC) is a highly malignant tumor of the head and neck region with a poor prognosis. Radiotherapy is one of the preferred treatment methods and remains irreplaceable. Despite continuous advancements in radiotherapy techniques, 35%–40% of patients still experience recurrence and develop radioresistance. Currently, the reasons for radioresistance in HSCC are unclear, and there are no specific methods to reduce it.

[0003] In the early stages of the study, the applicant discovered through proteomic sequencing of primary and recurrent tumor samples from patients with recurrent HSCC after radiotherapy that downregulation of SHMT1 (serine hydroxymethyltransferase 1) is of great significance for radiotherapy resistance in HSCC. As a key enzyme in one-carbon metabolism, SHMT1 is closely related to the occurrence and development of cancer. Based on this, the present invention is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide SHMT1 and its application in radiotherapy for head and neck tumors, in order to solve the problem that existing methods cannot reduce radiotherapy resistance.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an SHMT1 enzyme, wherein the amino acid sequence of the SHMT1 enzyme is shown in SEQ ID NO.1.

[0006] A polynucleotide that encodes an enzyme such as SHMT1.

[0007] A recombinant expression vector comprising polynucleotides.

[0008] Preferably, the recombinant expression vector includes either a cloning vector or an expression vector.

[0009] A transformant comprising a polynucleotide or a recombinant expression vector, wherein the transformant is not an animal or plant species.

[0010] A biological agent comprising the SHMT1 enzyme with the amino acid sequence shown in SEQ ID NO.1.

[0011] An enzyme composition comprising the SHMT1 enzyme.

[0012] The application of SHMT1 enzyme or enzyme combination in inhibiting radiotherapy resistance during HSCC radiotherapy.

[0013] Compared with existing technologies, the SHMT1 and its application in head and neck tumor radiotherapy provided by this invention, based on the unique regulatory role of SHMT1 in radioresistant hypopharyngeal squamous cell carcinoma, reveals for the first time a novel mechanism by which low expression of SHMT1 enhances tumor stem cell characteristics by activating GLI1 and downstream stemness-related genes, thereby promoting DNA damage repair and radiotherapy resistance. Experiments have demonstrated that knocking down SHMT1 reduces tumor sensitivity to radiotherapy, while overexpression of SHMT1 effectively inhibits tumor growth in vitro and in vivo. This indicates that SHMT1 is not only a key biomarker for predicting the prognosis of HSCC radiotherapy, but also a highly promising intervention target. The SHMT1 expression regulators provided by this invention can be used to prepare pharmaceutical compositions that improve radiotherapy efficacy and reduce the risk of recurrence, or to screen candidate compounds for reversing radiotherapy resistance. Simultaneously, it lays an experimental foundation for developing combined treatment strategies based on one-carbon metabolism and tumor stemness interaction, and is expected to significantly improve the long-term survival and quality of life of HSCC patients. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0015] Figure 1 A is a screening flowchart for 3 patients with recurrent HSCC tumors provided in an embodiment of the present invention. Figure 1 B is a volcano diagram of differentially expressed proteins in proteomics sequencing provided in an embodiment of the present invention. Figure 1 C is a bubble diagram representing the biological process analysis of differentially expressed proteins screened by proteomics according to an embodiment of the present invention. Figure 1 D is a bubble diagram representing the cellular component analysis of differentially expressed proteins screened by proteomics according to embodiments of the present invention. Figure 1 E is a bubble diagram representing the molecular functional analysis of differentially expressed proteins screened by proteomics according to embodiments of the present invention. Figure 1 F is a bubble chart representing the KEGG (Kyoto Encyclopedia of Genes and Genomes) signaling pathway analysis of differentially expressed proteins screened by the proteome according to embodiments of the present invention. Figure 1 G represents the bubble diagram in the KEGG signal pathway analysis. Figure 1 H is a KEGG pathway enrichment map provided in an embodiment of the present invention. Figure 1I is a GO (Gene Ontology) enrichment bubble chart provided in an embodiment of the present invention;

[0016] Figure 2 A represents a Venn diagram of genes corresponding to differentially expressed proteins detected in the TCGA (The Cancer Genome Atlas) database and proteomics, based on different gene expression methods provided in this embodiment of the invention. Figure 2 B represents the expression profiles of differentially expressed genes in FaDu and FR cells (experimentally constructed radioresistant FaDu cells) provided in the embodiments of the present invention. Figure 2 Figure C shows the expression of SHMT1 protein in FaDu and FR cells according to an embodiment of the present invention. Figure 2 D is a graph showing the immunohistochemical analysis results of SHMT1 in primary and recurrent tumor tissues after radiotherapy, provided in an embodiment of the present invention. Figure 2 E is a diagram showing the expression differences of exon SHMT1-related genes in FaDu and FR cells provided in the embodiments of the present invention. Figure 2 F is a graph showing the expression level of SHMT1 protein in HNSCC (Head and neck squamous cell carcinoma) provided in the embodiments of the present invention. Figure 2 G is a patient survival rate graph showing the difference in SHMT1 protein expression levels in tumors provided in the embodiments of the present invention;

[0017] Figure 3 Figure A shows the establishment of the SHMT1 knockdown cell line verified by qPCR according to an embodiment of the present invention. Figure 3 B is a diagram showing the establishment of SHMT1 knockdown cell lines as verified by Western blot analysis in an embodiment of the present invention. Figure 3 C is a graph showing the clonogenic capacity of SHMT1 knockdown Fadu cells before and after radiotherapy, provided in an embodiment of the present invention. Figure 3 D is a graph showing the proliferation capacity of Fadu cells before and after radiotherapy under SHMT1 knockdown, as provided in an embodiment of the present invention. Figure 3 E is a comet-like experimental diagram of SHMT1 knockdown Fadu cells after radiotherapy, provided in an embodiment of the present invention. Figure 3 F is a detection graph of DNA repair in SHMT1 knockdown Fadu cells before and after radiotherapy, provided in an embodiment of the present invention. Figure 3 G is a diagram showing the establishment of the SHMT1 overexpression cell line verified by qPCR according to an embodiment of the present invention. Figure 3 H is a diagram showing the establishment of SHMT1 overexpressing cell lines as verified by Western blotting in an embodiment of the present invention. Figure 3 Figure I is a graph showing the detection of clonogenic ability of SHMT1-overexpressing Fadu cells before and after radiotherapy, provided in an embodiment of the present invention. Figure 3 J is a detection diagram of DNA repair before and after radiotherapy in SHMT1-overexpressing Fadu cells provided in an embodiment of the present invention;

[0018] Figure 4 A is a diagram of the nude mouse radiotherapy experimental protocol provided in an embodiment of the present invention. Figure 4 B is a graph showing the monitoring and analysis of tumor growth dynamics provided in an embodiment of the present invention. Figure 4 C is a comparative analysis of the volume (left image) and weight (right image) of the isolated tumor provided in an embodiment of the present invention. Figure 4 D is an anatomical comparison diagram of mouse liver provided in the embodiments of the present invention;

[0019] Figure 5 A is a performance graph of differentially expressed genes provided in an embodiment of the present invention. Figure 5 B is a graph showing the up-regulation and down-regulation of gene expression provided in an embodiment of the present invention. Figure 5 C is a genetic difference analysis diagram provided in an embodiment of the present invention. Figure 5 D is a graph showing the enrichment analysis of differentially expressed genes in GO-related pathways provided in an embodiment of the present invention. Figure 5 E is a graph showing the enrichment analysis of differentially expressed genes in KEGG-related pathways provided in an embodiment of the present invention. Figure 5 F is a qPCR verification diagram provided in this embodiment of the invention, confirming that SHMT1 knockdown increases GLI1 expression. Figure 5 G is a graph showing the increase in expression of tumor stem cell markers verified by qPCR in an embodiment of the present invention. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0021] As attached Figure 1 To be continued Figure 5 As shown:

[0022] Example:

[0023] An SHMT1 enzyme, the amino acid sequence of which is shown in SEQ ID NO.1.

[0024] SEQ ID NO.1

[0025] MTMPVNGAHKDADLWSSHDKMLAQPLKDSDVEVYNIIKKESNRQRVGLELIASENFASRA

[0026] VLEALGSCLNNKYSEGYPGQRYYGGTEFIDELETLCQKRALQAYKLDPQCWGVNVQPYSG

[0027] SPANFAVYTALVEPHGRIMGLDLPDGGHLTHGFMTDKKKISATSIFFESMPYKVNPDTGY

[0028] INYDQLEENARLFHPKLIIAGTSCYSRNLEYARLRKIADENGAYLMADMAHISGLVAAGV

[0029] VPSPFEHCHVVTTTTHKTLRGCRAGMIFYRKGVKSVDPKTGKEILYNLESLINSAVFPGL

[0030] QGGPHNHAIAGVAVALKQAMTLEFKVYQHQVVANCRALSEALTELGYKIVTGGSDNHLIL

[0031] VDLRSKGTDGGRAEKVLEACSIACNKNTCPGDRSALRPSGLRLGTPALTSRGLLEKDFQK

[0032] VAHFIHRGIELTLQIQSDTGVRATLKEFKERLAGDKYQAAVQALREEVESFASLFPLPGL

[0033] A polynucleotide that encodes an enzyme such as SHMT1.

[0034] A recombinant expression vector comprising polynucleotides.

[0035] The recombinant expression vector includes either a cloning vector or an expression vector.

[0036] A transformant comprising a polynucleotide or a recombinant expression vector, wherein the transformant is not an animal or plant species.

[0037] A biological agent comprising the SHMT1 enzyme with the amino acid sequence shown in SEQ ID NO.1.

[0038] An enzyme composition comprising the SHMT1 enzyme.

[0039] The application of SHMT1 enzyme or enzyme combination in inhibiting radiotherapy resistance during HSCC radiotherapy.

[0040] Based on the unique regulatory role of SHMT1 in radioresistant hypopharyngeal squamous cell carcinoma (HSCC), this study reveals a novel mechanism by which low SHMT1 expression enhances tumor stem cell characteristics through activation of GLI1 (Glioma-associated oncogene family member 1) and downstream stemness-related genes, thereby promoting DNA damage repair and radioresistance. Experiments demonstrate that knocking down SHMT1 reduces tumor sensitivity to radiotherapy, while overexpression effectively inhibits tumor growth in vitro and in vivo. This indicates that SHMT1 is not only a key biomarker for predicting the prognosis of HSCC radiotherapy but also a highly promising intervention target. The SHMT1 expression regulators provided in this invention can be used to prepare pharmaceutical compositions that improve radiotherapy efficacy and reduce recurrence risk, or to screen candidate compounds for reversing radioresistance. Furthermore, this study lays an experimental foundation for developing combined treatment strategies based on one-carbon metabolism and tumor stemness interaction, potentially significantly improving the long-term survival and quality of life of HSCC patients.

[0041] SHMT1 gene was knocked down and overexpressed in HSCC cell lines, followed by 4 Gy irradiation. Cell colony formation assays and CCK8 assays were performed after irradiation to verify cell proliferation at different SHMT1 gene expression levels. Immunofluorescence assays were also used to verify changes in γ-H2AX in cells with different expression levels, observing DNA damage repair. Furthermore, SHMT1 knockdown cell tumor-bearing mice were used to verify the effect of SHMT1 expression levels on radiotherapy in vivo. Finally, transcriptomics analysis was used to identify downstream genes of GLI1.

[0042] Analysis of the UALCAN database (University of Alabama at Birmingham Cancer Database) revealed differential SHMT1 expression between HNSCC and adjacent normal cells, and a significant correlation with patient prognosis. In clonogenic assays, SHMT1-expressing cells showed significantly weaker proliferation than the control group both before and after radiotherapy, while SHMT1-knockdown cells showed significantly stronger proliferation. Similar findings were observed in the CCK8 assay. Similar results were also observed in mice, where SHMT1-knockdown tumors were larger than the control group both before and after irradiation. Immunofluorescence analysis of γ-H2AX expression showed that after 6 Gy X-ray irradiation, SHMT1-knockdown cells exhibited significantly less DNA damage than the control group, while SHMT1-overexpressing cells showed significantly more DNA damage. Transcriptome sequencing analysis revealed significantly increased GLI1 expression in SHMT1-knockdown cells. GLI1 is a classic Hedgehog pathway-related gene, associated with tumor stemness. Further qPCR validation of tumor stemness-related genes SOX9, SOX2, CD133, CD44, CD47, EPCAM, ALDH1, CMYC, and NANOG revealed that the expression levels of these genes were all elevated.

[0043] SHMT1, a low-expressed gene associated with radiotherapy resistance in HSCC, is downregulated by promoting DNA repair and tumor cell proliferation before and after radiotherapy, leading to radiotherapy resistance and consequently poor patient prognosis. Our experiments further confirmed the association between SHMT1 and radiotherapy resistance in HSCC and explored the downstream Hedgehog pathway-related gene GLI1, demonstrating that the occurrence of radiotherapy resistance in HSCC is related to tumor stemness.

[0044] Screening for differentially expressed proteins and genes between postoperative radiotherapy-induced recurrent HSCC tumor tissue and primary tumor tissue:

[0045] After screening, three HSCC tumor patients meeting the criteria were obtained (Figure 1A). Proteomics sequencing of the three postoperative radiotherapy-recurrent HSCC tumor tissues revealed 2791 quantifiable proteins. Using the primary tumor tissue as a control, 52 differentially expressed proteins were screened, with 20 upregulated and 32 downregulated (Figure 1B). GO analysis showed that, in terms of biological processes, the differentially expressed proteins were enriched in fatty acid derivative metabolism (Figure 1C); in terms of cellular composition, they were enriched in immunoglobulin complexes, high-density lipoprotein microparticles, and lipoprotein microparticles (Figure 1D); and in terms of molecular function, they were enriched in isomerase activity, ligase activity, and carbon-sulfur bond formation (Figure 1E). KEGG analysis showed that the differentially expressed proteins were mostly enriched in metabolic pathways (Figure 1F). Data from 500 HSCC tumor tissue samples and 44 normal tissue samples were downloaded from the TCGA database. Using the normal tissue as a control, a total of 5033 differentially expressed genes were screened, with 2763 upregulated and 2270 downregulated. The specific expression is shown in the volcano diagram (Figure 1G). GO enrichment analysis results show that, in terms of biological processes, differentially regulated genes are mainly enriched in muscle system processes; in terms of cellular composition, differentially regulated genes are mainly enriched in collagen-containing extracellular matrix and immunoglobulin complexes; and in terms of molecular function, differentially regulated genes are mainly enriched in antigen binding and immunoglobulin receptor binding (Figure 1H). KEGG enrichment analysis results show that upregulated differentially regulated genes are mainly enriched in the PI3K / Akt signaling pathway, small cell lung cancer, cytokine-cytokine receptor interaction, and cell cycle pathways; downregulated differentially regulated genes are mainly enriched in fatty acid degradation, tyrosine metabolism, and the PPAR signaling pathway (Figure 1I).

[0046] SHMT1 expression levels in postoperative radiotherapy-recurrent HSCC tumor tissue were lower than in primary tumor tissue.

[0047] Differentially expressed genes screened from the TCGA database in HNSCC were compared with the coding genes of differentially expressed proteins obtained from proteomics sequencing, revealing six overlapping genes (Figure 2A). Cellular-level qPCR validation of these six overlapping genes showed that, compared to FaDu cells, five genes, including SHMT1, were downregulated in FR cells (P < 0.001), while CES2 expression showed no difference between FaDu and FR cells (Figure 2B). Proteomics results showed that, compared to primary tumor tissue, ABLIM2, PTK7, LAMB3, NEXN, and CES2 were all upregulated in tumor tissue recurring after postoperative radiotherapy, showing the opposite trend to the cellular level. The trend of SHMT1 gene expression at the cellular level was consistent with that of proteomics. Therefore, SHMT1 was selected as the final research gene. Western blot experiments showed that, compared to FaDu cells, the expression level of SHMT1 protein in FR cells was significantly decreased (Figure 2C, P < 0.001). To further confirm the downregulation of SHMT1 expression in radiotherapy resistance, immunohistochemistry was used to detect the SHMT1 protein expression level in three patients. The results showed that SHMT1 expression was reduced in recurrent tumor tissue after postoperative radiotherapy compared to the primary tumor (Figure 2D). In FR and FaDu cells, qPCR validation of other related genes in the one-carbon metabolism pathway involved in SHMT1 revealed that the expression of one-carbon metabolism-related factors was decreased in FR cells (P < 0.05), consistent with changes in SHMT1 expression (Figure 2E). SHMT1 is a key gene in one-carbon metabolism, catalyzing the conversion of serine to glycine and tetrahydrofolate. One-carbon metabolism is involved in biosynthesis (purines and thymidine), amino acid homeostasis (glycine, serine, and methionine), maintenance of epigenetic status, and redox reactions. Furthermore, both genetic and functional evidence suggest that excessive activation of one-carbon metabolism can lead to tumorigenesis. Multiple studies and databases have mentioned that SHMT1 is highly expressed in various tumors and participates in cancer development and progression. However, SHMT1 appeared to play a different role than usual during the radiotherapy of hypopharyngeal squamous cell carcinoma in our study. To further verify the role of SHMT1 in head and neck squamous cell carcinoma, we used the UALCAN database and found that the expression level of SHMT1 in the primary tumor was significantly higher than that in adjacent normal tissue. Figure 2 F). Survival analysis using the UALCAN database showed that higher SHMT1 expression was associated with shorter survival in patients with lenticular squamous cell carcinoma (Fig. 2G), suggesting its involvement in the development of head and neck squamous cell carcinoma and the potential for targeting it for radiotherapy-resistant HNSCC. In conclusion, these data indicate that SHMT1 is associated with poor prognosis in HNSCC patients, suggesting that SHMT1-targeted therapy has great potential as an effective treatment strategy for radiotherapy-resistant HNSCC.

[0048] Establish SHMT1 knockdown and overexpression cell lines to verify the differences in cell radiotherapy:

[0049] Stable SHMT1 knockdown FaDu cells were generated using lentiviruses, and the knockdown efficiency was verified using qPCR (Figure 3A) and Western blotting (Figure 3B). Colony formation assays revealed that the number of colonies and the area of ​​individual colonies in untreated FaDu cells were significantly higher than in the control group, and this result remained unchanged after 4 Gy radiotherapy (Figure 3C). Reduced SHMT1 expression negated the therapeutic effect of radiotherapy in FaDu cells. This result was further confirmed in CCK8 cell proliferation experiments: regardless of whether 6 Gy radiation was received, the cell proliferation rate in the shRNA group was faster than that in the control group. Notably, both groups of untreated cells reached peak growth around day 6 of culture; thereafter, we hypothesize that high cell density may have led to contact inhibition or nutrient depletion, resulting in a significant decrease in cell viability. Figure 3 D). Therefore, these data indicate that SHMT1 is a key factor in inducing radiotherapy resistance in HNSCC. We further used comet assays and immunofluorescence to detect the expression of γ-H2AX, a marker of DNA double-strand breaks, to assess the cells' ability to repair radiotherapy-induced DNA damage. We found that there was no significant difference in DNA damage capacity between cells knocked down before radiotherapy and the control group, but after radiotherapy, SHMT1 knockdown cells showed significantly weaker DNA damage capacity than the control group (D). Figure 3 E, F). Furthermore, we prepared FaDu cells transiently overexpressing SHMT1 (SHMT1+ / +) using plasmids and analyzed the results using qPCR (…). Figure 3 G) and WB ( Figure 3 H) Verify SHMT1 overexpression efficiency. Clonogenesis revealed that SHMT1-overexpressing cells were significantly more sensitive to radiotherapy than the control group (H). Figure 3 I). Using immunofluorescence, we found that before radiotherapy, there was no significant difference in DNA damage capacity between the overexpressing cells and the control group, but after radiotherapy, the DNA damage in SHMT1-overexpressing cells was significantly more severe than that in the control group. Figure 3 J).

[0050] In vivo validation of the radioresistance of SHMT1 knockdown cell lines:

[0051] To further validate these results in vivo, we constructed a xenograft tumor nude mouse model (Figure 4A). Tumor tissue data showed that knocking down SHMT1 significantly enhanced tumor growth and weakened the therapeutic effect of radiotherapy (Figure 4B), and tumor growth curves showed similar results (Figure 4C). We regularly monitored the weight of the nude mice and found that both groups of mice receiving radiotherapy experienced a brief decrease in weight at the beginning of radiation exposure, which then gradually recovered. By the end of the experiment, the weights of the four groups of mice were similar, and no significant statistical differences were observed, indicating that radiation therapy did not cause significant systemic toxicity (Figure 4D).

[0052] Screening for differentially expressed genes between SHMT1 knockdown of Fadu and control group Fadu:

[0053] Transcriptomic sequencing was performed on SHMT1 knockdown Fadu and the control group Fadu. The differentially expressed gene heatmap showed upregulated and downregulated genes in the shRNA group, while the control group exhibited the opposite trend (Figure 5A). Transcriptomic sequencing analysis identified 1209 differentially expressed genes, including 739 upregulated genes and 470 downregulated genes (Figure 5B). Venn plot analysis revealed 10826 co-expressed genes between the shRNA and control groups, and 589 and 369 unique differentially expressed genes, respectively, in the shRNA and control groups (Figure 5C). GO functional enrichment analysis of these differentially expressed genes indicated that their molecular functions mainly involve molecular function regulators and signaling receptor binding, and their cellular components are primarily extracellular regions (Figure 5D). KEGG signaling pathway enrichment analysis showed that these differentially expressed genes were significantly enriched in pathways such as mineral absorption, cytokine-cytokinereceptor interaction, and the TNF signaling pathway (Figure 5E). To clarify the molecular mechanism of SHMT1-induced changes in the radiosensitivity of HSCC cells, we further screened differentially expressed genes and identified upregulated expression of GLI1, a gene closely related to tumor stem cells (Figure 5F). Further qPCR validation of tumor stemness-related genes such as SOX9, SOX2, CD133, CD44, CD47, EPCAM, ALDH1, CMYC, and NONAG revealed that all of these genes were upregulated (Figure 5G).

[0054] Materials and Methods:

[0055] 1. Clinical specimen collection

[0056] We screened for recurrent cases that met the objectives of this study using the following criteria: (1) two surgical pathologies confirmed HSCC; (2) a single tumor; (3) no prior radiotherapy, chemotherapy, or other treatments were received before the primary tumor treatment; and (4) the primary tumor underwent radical treatment with surgery and radiotherapy. Ultimately, we screened three cases with primary tumor tissue and tumor tissue samples from recurrent tumors after postoperative radiotherapy.

[0057] 2. Cell Culture

[0058] The hypopharyngeal carcinoma cell line FaDu was cultured routinely in a cell culture incubator at 37°C and 5% CO2 saturated humidity. The complete culture medium used for cell growth consisted of 90% MEM basal medium (TBD), 10% fetal bovine serum, and 1% penicillin-streptomycin solution. All cell manipulations were performed in a laminar flow hood irradiated with ultraviolet light for 30 minutes, and the work surface and laboratory equipment were sterilized with 75% alcohol.

[0059] 3. Lentiviral transfection

[0060] To stably knock down SHMT1 expression, sequence-specific shRNA was used. Cells were transfected with polyberen (Invitrogen, CA, USA) (10 μg / ml) in culture medium, using either the targeting shRNA with a viral titer of 1*10^8 or a negative control shRNA (GenePharma, Shanghai, China). After 24 hours of transfection, the culture medium was replaced with fresh medium. Cells were incubated for another 48 hours, and selection was performed using Puromycin. Further experiments were conducted after verifying the knockdown efficiency of the target gene using qPCR.

[0061] 4. Plasmid extraction

[0062] Bacteria were collected from a -80℃ glycerol culture and inoculated into LB medium containing ampicillin. The culture was activated and amplified in a shaker at 37℃. After centrifugation to collect the bacterial cells, a commercial plasmid extraction kit was used. The resulting plasmid DNA solution was analyzed for concentration and purity, then aliquoted and stored at -20℃ for later use.

[0063] 5. Plasmid transfection

[0064] Transient overexpression of SHMT1 was performed using p3000 enhancer and Lipo3000 transfection reagent in the culture medium, followed by the addition of extracted siRNA and negative control cells. After 24 hours of transfection, the culture medium was replaced with fresh medium, and cells were cultured for another 24–48 hours before being collected for further analysis.

[0065] 6. CCK8 determination

[0066] Fadu cells (1 × 10^3) ​​were placed in 96-well plates and incubated for 24 hours before being radiotreated with a dose of 6 Gy. Cell viability was measured every 24 hours. For measurement, 10 μL of CCK-8 solution was added to each well and incubated for 2 hours. Absorbance was measured at 450 nm using a microplate reader.

[0067] 7. Cloning

[0068] Cells were seeded into 6-well plates (1,000 cells / well) and incubated for 24 hours before radiotherapy, with the culture medium changed every 48 hours. After 10–14 days of incubation, colonies were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The colony count for each treatment group was calculated in three replicate wells.

[0069] 8. RNA isolation, reverse transcription, and quantitative PCR (qPCR)

[0070] Total RNA was extracted from cells using Freezol Reagent and reverse transcribed to cDNA using SPARKscript II All-in-oneRT SuperMix (qPCR with gDNA eraser). qPCR was performed using ChamQ SYBR qPCR premix (Vazyme) following the manufacturer's cycling and temperature instructions. Gene expression was normalized using β-ACTIN as an internal control. The following primer sequences were used:

[0071] SHMT1-forward GCTACGGAAGATTGCAGATGAGAAC

[0072] SHMT1-reverse GGTCTTGTGAGTGGTGGTGGTC

[0073] ACTIN-forward TGACGTGGACATCCGCAAAG

[0074] ACTIN-reverse CTGGAAGGTGGACAGCGAGG

[0075] GLI1-forward CCTTGTGGTCCCCATGACTC

[0076] GLI1-reverse AGGGGATCCTGTATGCCTGT

[0077] SOX9-forward AGCTGTGTGTAGACGGGTTG

[0078] SOX9-reverse TTCACCTACATGAACCCCGC

[0079] SOX2-forward TGTGCATCTTGGGGTTCTCC

[0080] SOX2-reverse CAACCAGAAAAACAGCCCGG

[0081] CD133-forward GAAGGACTCGTTGCTGGTGA

[0082] CD133-reverse AACAGTTTGCCCCCAGGAAA

[0083] CD44-forward GCAGGGATTCTGTCTGTGCT

[0084] CD44-reverse CTCCAGTGAAAGGAGCAGCA

[0085] CD47-forward ACGCCGCAATACAGAGACTC

[0086] CD47-reverse AACCTCCTTCGTCATTGCCA

[0087] EPCAM-forward ATCATTGTTCTGGAGGGCCC

[0088] EPCAM-reverse AGCTGGCTGCCAAATGTTTG

[0089] ALDH1-forward AACACTGTGGGCTGGACAAA

[0090] ALDH1-reverse ATCCTCTGACCCCAGGAGTC

[0091] CMYC-forward GCTGGTGCATTTTCGGTTGT

[0092] CMYC-reverse CCCTCCACTCGGAAGGACTA

[0093] NANOG-forward TCTGCTGGAGGCTGAGGTAT

[0094] NANOG-reverse ACCAGTCCCAAAGGCAAACA

[0095] 9. Western blot

[0096] Cell and tissue proteins were extracted using RIPA lysis buffer (SparkJade) containing 1% PMSF, PIA, and PIB. Separation was performed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 80 V for 30 min, followed by separation at 120 V for 60 min, and then transferred to a PVDF membrane. The membrane was blocked at room temperature for 2 h, then incubated overnight with primary antibody (Sangon Biotech) at 4 °C. Subsequently, the membrane was incubated with secondary antibody (Sangon Biotech) at room temperature for 2 h.

[0097] 10. Plasmids, antibodies, and lentiviruses

[0098] SHMT1 was cloned into the pcDNA3.1 vector. SHMT1 and its precursor were prepared using the KOD-Plus mutagenesis kit according to the manufacturer's instructions. Cells were transfected with Lipofectamine 3000 (L3000075, Thermo Fisher Scientific) according to the provided instructions. Chemical intervention and protein assays were performed 48 hours post-transfection.

[0099] 11. Immunofluorescence

[0100] Fadu cells transfected with lentivirus were cultured on glass coverslips and cultured after 6 Gy radiotherapy, with pauses at 30 min, 1 h, 6 h, and 12 h post-radiotherapy. Cells were fixed in 4% paraformaldehyde for 20 min and blocked with 5% BSA for 1 h. Subsequently, cells were incubated overnight at 4°C with the designated primary antibody, followed by treatment with a fluorescently conjugated secondary antibody for 1 h. Cell nuclei were counterstained with DAPI for 10 min, and images were captured using a fluorescence microscope.

[0101] 12. Xenograft Tumor Model

[0102] By using 0.1 ml of 10 7 A xenograft tumor model was established by subcutaneously injecting SHMT1-knockdown Fadu cells and control Fadu into the right axillary region of 5-week-old male BALB / c nude mice. When the tumor reached approximately 0.5 cm³, the mice were randomly divided into four groups and subjected to radiotherapy at a regimen of 5 Gy per day for 3 consecutive days. Mouse weight and tumor volume were measured daily, and the mice were observed for 30 days. Mice were then sacrificed, and the xenograft tumors were excised for statistical analysis. All mice were housed in a specific pathogen-free environment in the animal room of Yantai Yuhuangding Hospital. All mouse experiments followed the guidelines of the Institutional Animal Care and Use Committee and were approved by the Medical Ethics Committee of Yantai Yuhuangding Hospital.

[0103] 13. Transcriptome sequencing and bioinformatics analysis

[0104] Fadu cells were cultured separately for the control group and the SHMT1 knockdown group, with three independent biological replicates for each group. Cell pellets were collected, lysed with Freezol Reagent, and then flash-frozen in liquid nitrogen. All samples were sequenced by Beijing Novogene Technology Co., Ltd. 150 bp paired-end sequencing was performed using the Illumina NovaSeq 6000 platform. Data were quality controlled and filtered using Fastp software. High-quality reads were aligned to the human reference genome GRCh38.p13 using HISAT2. Gene counting was performed using featureCounts, and differentially expressed genes were analyzed using the DESeq2 software package in the R environment. The selection criteria were: corrected P-value (Padj) < 0.05 and |log2 (fold change)| ≥ 1.

[0105] 14. Statistical Analysis

[0106] All experimental data were derived from at least three independent biological replicates and are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism 9.5 software. Unpaired t-tests were used for comparisons between two groups; one-way ANOVA was used for comparisons between three or more groups, with multiple comparison tests performed. A p-value less than 0.05 was considered statistically significant.

[0107] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A SHMT1 enzyme, characterized in that, The amino acid sequence of the SHMT1 enzyme is shown in SEQ ID NO.

1.

2. A polynucleotide, comprising, The polynucleotide encodes the SHMT1 enzyme as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide as described in claim 2.

4. The recombinant expression vector of claim 3, wherein, The recombinant expression vector includes either a cloning vector or an expression vector.

5. A transformant characterized in that, The transformant comprises the polynucleotide as described in claim 2, or the recombinant expression vector as described in claims 3-4, wherein the transformant is not an animal or plant species.

6. A biological agent, characterized in that, The enzyme contains the SHMT1 enzyme with the amino acid sequence shown in SEQ ID NO.

1.

7. An enzyme composition, characterized in that, The enzyme composition comprises the SHMT1 enzyme as described in claim 1.

8. The application of the SHMT1 enzyme as described in claim 1 or the enzyme composition as described in claim 7 in inhibiting radioresistance in HSCC radiotherapy.

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