Application of gene PTHLH as target point in auxiliary diagnosis, prognosis judgment and treatment of esophageal squamous carcinoma

Abstract

The application belongs to the technical field of biological medicine, and provides application of gene PTHLH as a target point in auxiliary diagnosis, prognosis judgment and treatment of esophageal squamous carcinoma. The target point PTHLH is an esophageal squamous carcinoma driving target point accumulated based on structural variation TD, that is, a potential target point of a super enhancer accumulated by TD; the super enhancer is an upstream regulation region of PTHLH. The application research finds that the enhancer region of the ESCC driving gene is frequently amplified by TD. These results suggest that the TD event can regulate the related expression of the cancer gene by affecting the gene ontology and the regulation element. Meanwhile, the esophageal squamous carcinoma driving target point accumulated based on the structural variation TD is found to be the potential target point PTHLH of the super enhancer accumulated by TD.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of gene PTHLH as a target point in auxiliary diagnosis, prognosis judgment and treatment of esophageal squamous cell carcinoma, and further, application of PTHLH as a target point of esophageal squamous cell carcinoma in auxiliary diagnosis, prognosis judgment and treatment of esophageal squamous cell carcinoma driven by structural variation of tandem duplication (TD). BACKGROUND

[0002] Esophageal cancer is one of the most common malignant tumors in the digestive system, and its incidence and mortality rates are among the highest in the world. There are two main histological types of esophageal cancer: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). Among them, ESCC is the main histological type in China, and its incidence and mortality rates are the highest compared with other countries. In recent years, although technical progress has been made in diagnosis and treatment, the prognosis of the disease is still poor due to late diagnosis and lack of effective target points. Therefore, exploring the pathogenesis and potential therapeutic targets of ESCC and promoting the personalized treatment of ESCC have become an important task at this stage.

[0003] In the past decade, researchers have intensively explored potential driver targets in ESCC from different perspectives, such as different populations, multi-site sequencing and omics integration analysis. Based on genome sequencing, these studies have explored significant mutant genes that play an important role in the tumorigenesis of ESCC, including NOTCH1, ZNF750, NFE2L2 and PIK3CA, etc. However, in ESCC, not only significant gene mutations are involved, but also a large number of structural variations (SVs) exist, which may involve more driver genes and have a more profound impact on gene function and tumor development.

[0004] According to the results of previous studies, multiple structural variation types including deletion, inversion and tandem duplication (TD) were detected, which frequently occur in ESCC and involve multiple potential driver genes, including cancer genes such as KLF5 and ERBB2, and tumor suppressor genes such as CDKN2A and FAT1. In addition, structural variations not only involve the gene body region, but also affect the copy number expansion of the regulatory region (such as enhancer).

[0005] PTHLH is a parathyroid hormone-like hormone, the gene encodes a protein that is a member of the parathyroid hormone family, which regulates intramembranous bone development in the process of mammary gland and tooth formation and epithelial-mesenchymal interaction through its receptor PTHR1. It is also an important regulator of organ growth, development, migration, differentiation and epithelial calcium transport, which is essential for bone homeostasis. PTHLH mainly interacts with the parathyroid hormone / parathyroid hormone-related protein receptor (PTH / PTHLH-1 receptor, PTH1R) member of the B group G protein-coupled receptor, which controls various cell functions by activating the cAMP / PKA or IP3 / PKC signal cascade.

[0006] In published tumor studies, PTHLH gene plays an important role in various cancers. The expression protein PTHrP of PTHLH is particularly important for the development of bone and breast, which plays a role in paracrine and endocrine, and is also highly expressed in more than two-thirds of breast tumor tissue samples. It is believed to affect the proliferation and migration of breast cancer cells, and a large number of literature has confirmed that PTHrP protein is a promoting factor for breast cancer bone metastasis, which is mainly attributed to TGFb-driven PTHrP paracrine and subsequent osteolysis and hypercalcemia, and high levels of PTHrP protein in primary tumors are also associated with an increased risk of bone metastasis. PTHLH also exists as a susceptibility gene for triple-negative breast cancer. In colorectal cancer, PTHLH also participates in important functional regulation, researchers found that overexpression of PTHrP is associated with increased migration and invasion of colorectal cancer cells in vitro and in vivo. PTHrP can induce cell migration and regulate FAK protein expression through the ERK / RSK signaling pathway, and can also promote the migration and invasion ability of colorectal cancer cells through Rho-GTPase Rac1 mediation.

[0007] In combination with the results of bone metastasis in breast cancer, these studies show that PTHLH plays an important role in tumor metastasis. In addition, in studies including oral squamous cell carcinoma and head and neck squamous cell carcinoma, it was found that PTHLH can be used as a potential tumor marker, which may participate in the pathogenesis of squamous cell carcinoma by affecting cell proliferation and cell cycle. In addition, the PTHLH gene can be a potential new target for the treatment of pancreatic cancer, as an oncogene, it usually appears with KARS amplicon. Although there are related reports of PTHLH gene in esophageal cancer, it is only limited to finding that it is accompanied by increased expression and the presence of super enhancer in esophageal cancer, and its mechanism of action and function in esophageal cancer have not been further studied. SUMMARY

[0008] The application provides application of gene PTHLH as a target point in auxiliary diagnosis, prognosis judgment and treatment of esophageal squamous carcinoma, and further provides application of PTHLH as a target point of esophageal squamous carcinoma driven by structural variation of tandem duplication (TD) in auxiliary diagnosis, prognosis judgment and treatment of esophageal squamous carcinoma. The application finds that a potential driver gene PTHLH of a super-enhancer target point affected by structural variation TD in ESCC is significantly amplified in esophageal squamous carcinoma. The regulation of PTHLH by upstream enhancer amplification of PTHLH is determined. The relationship between PTHLH and prognosis and metastasis of patients with esophageal squamous carcinoma is determined. The expression regulation of PTHLH gene and the mechanism of PTHLH in occurrence and development of esophageal squamous carcinoma are clarified.

[0009] The application is realized by the following technical solutions: application of gene PTHLH as a target point in preparation of a drug for diagnosing esophageal squamous carcinoma, the target point PTHLH is a driving target point of esophageal squamous carcinoma accumulated based on structural variation TD, that is, a potential target point PTHLH of a super-enhancer accumulated by TD, the super-enhancer is an upstream regulation region of PTHLH, and the nucleotide sequence is as shown in the following: the nucleotide sequence of enhancer Enhancer e3 (hg19 chr12: chr12:28176001-28177500) is as shown in SEQ ID NO:1; the nucleotide sequence of enhancer Enhancer e5 (hg19 chr12: chr12:28185600-28186900) is as shown in SEQ ID NO:2; and the nucleotide sequence of enhancer Enhancer e8 (hg19 chr12: chr12:28284501-28285500) is as shown in SEQ ID NO:3.

[0010] The application also provides application of the gene PTHLH as a target point in preparation of a reagent for screening an anti-esophageal squamous carcinoma drug.

[0011] In addition, the application also provides: the application of gene PTHLH or super enhancer as a target point in preparation of an esophageal squamous carcinoma prognosis evaluation reagent, the target point PTHLH is an esophageal squamous carcinoma driving target point accumulated based on structural variation TD, that is, a potential target point PTHLH of a super enhancer accumulated by TD; and the super enhancer is an upstream regulation region of PTHLH, and the nucleotide sequence is as follows: the nucleotide sequence of enhancer Enhancer e3 (hg19 chr12: chr12:28176001-28177500) is shown in SEQ ID NO:1; the nucleotide sequence of enhancer Enhancer e5 (hg19 chr12: chr12:28185600-28186900) is shown in SEQ ID NO:2; and the nucleotide sequence of enhancer Enhancer e8 (hg19 chr12: chr12:28284501-28285500) is shown in SEQ ID NO:3.

[0012] The TD event affecting the PTHLH enhancer is determined by DNA sequencing.

[0013] The application uses whole genome sequencing to perform whole genome sequencing on 528 esophageal squamous carcinoma tumor tissues and paired normal tissues, uses the method of SvABA and Delly for the sequencing results, finds that a TD hot event exists in the upstream of the PTHLH gene, and uses the GISTIC method to find that the TD event can cause significant amplification of the upstream region of the PTHLH gene. Then, the TD event in the esophageal squamous carcinoma sample is verified by the method of q-PCR and sanger sequencing.

[0014] The application discloses that the correlation between the amplification of the upstream enhancer of PTHLH and the expression of PTHLH and the significant expression up-regulation of PTHLH in the esophageal squamous carcinoma tumor sample are analyzed by using student-t test, which indicates that the amplification of the upstream enhancer of PTHLH caused by the TD event in the esophageal squamous carcinoma can significantly up-regulate the expression of the PTHLH gene.

[0015] The method of Kaplan-Meier survival analysis and Fisher's exact test is used to analyze the relationship between PTHLH and the prognosis and metastasis of esophageal squamous carcinoma patients. The results show that PTHLH gene is significantly related to the survival prognosis and metastasis of patients.

[0016] The expression level of PTHLH in different esophageal squamous carcinoma cell lines is detected by Western blot, a cell strain with stable low expression of PTHLH is constructed, and the change of the phenotype of the esophageal squamous carcinoma cell after knocking down PTHLH is detected by MTT, hard cloning and flow cytometry experiments.

[0017] The application breaks the previous potential target research mainly based on mutation, and the potential target cancer gene PTHLH is screened from the structural variation level. Through large sample structural variation research, a hot TD event of PTHLH gene and its upstream is found, which significantly expands the super-enhancer region of the PTHLH gene and promotes the up-regulation of gene expression. The PTHLH gene is significantly related to the prognosis and metastasis of ESCC patients, and the preliminary functional experiment verifies the above conclusion. The expansion of the gene and its upstream enhancer can be used as a potential predictor of the prognosis and metastasis of ESCC patients. Targeted therapy for the hot spot can reduce the proliferation and metastasis of ESCC cells.

[0018] The application breaks the previous potential target research mainly based on mutation, and the potential target cancer gene PTHLH is screened from the structural variation level. Through large sample structural variation research, a hot TD event of PTHLH gene and its upstream is found, which significantly expands the super-enhancer region of the PTHLH gene and promotes the up-regulation of gene expression. The PTHLH gene is significantly related to the prognosis and metastasis of ESCC patients, and the preliminary functional experiment verifies the above conclusion. The expansion of the gene and its upstream enhancer can be used as a potential predictor of the prognosis and metastasis of ESCC patients. Targeted therapy for the hot spot can reduce the proliferation and metastasis of ESCC cells. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Fig. 1 is a copy number heat map result of PTHLH gene around chromosome 12 in 85 ESCC samples; the lower part of the figure includes chip-seq enhancer annotation and TAD boundary of esophageal cells and esophageal cancer cell lines.

[0020] Figure 2 Fig. 3 is the verification result of 11 TD events, the upper part is the q-PCR verification result figure, and the lower part is the sanger sequencing verification result.

[0021] Figure 3 Fig. 4 is the dual luciferase reporter experiment result of enhancer verification, the left side is the signal intensity column chart of e1-e8 sequence verification, and the right side is the signal intensity column chart of e3 and e5 sequence refinement, *P≤0.05, **P≤0.01.

[0022] Figure 4 Fig. 5 is the amplification and expression up-regulation of PTHLH gene. A: The correlation box plot of enhancer and gene amplification and RNA expression. B: The box plot of PTHLH RNA expression in tumor and paracancerous control. C: The box plot of PTHLH protein expression in tumor and paracancerous control.

[0023] Figure 5 Fig. 6 is the clinical correlation analysis result of PTHLH gene, including survival analysis and metastasis correlation analysis. A: The correlation of PTHLH amplification and ESCC prognosis and metastasis. B: The correlation of PTHLH expression and ESCC prognosis and metastasis.

[0024] Figure 6Figure for cell function experiment results of PTHLH gene in ESCC cell line KYSE450. Upper left corner: bar chart shows the knock-out efficiency of PTHLH. Upper right corner: line chart shows the cell proliferation after knocking out PTHLH by MTT method, and bar chart shows the colony number in cells. Lower left: cell migration was monitored by scratch test, and bar chart shows the migration results. Lower right: Transwell cell migration and invasion analysis after PTHLH knock-out.

[0025] Figure 7 Figure for cell function experiment results of PTHLH gene in ESCC cell line KYSE180. Upper left corner: bar chart shows the knock-out efficiency of PTHLH. Upper right corner: line chart shows the cell proliferation after knocking out PTHLH by MTT method, and bar chart shows the colony number in cells. Lower left: cell migration was monitored by scratch test, and bar chart shows the migration results. Lower right: Transwell cell migration and invasion analysis after PTHLH knock-out. DETAILED DESCRIPTION

[0026] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work, fall within the protection scope of the present application.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, and the materials cited and referenced herein are incorporated by reference in their entirety.

[0028] All equivalents of the described specific implementations that would be obvious to those skilled in the art and that are within the scope of the present application are intended to be within the scope of the claims.

[0029] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0030] I. Experimental materials

[0031] 1. Study subjects: Tumor and paired adjacent normal tissue samples were collected from 528 patients with esophageal squamous cell carcinoma (ESCC). All patients did not receive chemotherapy or radiotherapy before surgery. All subjects gave informed consent, and the study was approved by the Institutional Review Board of Shanxi Medical University and Shanxi Tumor Hospital (Shanxi, China). At least three independent pathologists reviewed the H&E-stained sections of each sample to confirm that the tumor specimen was histologically consistent with ESCC and that the adjacent normal specimen did not contain tumor cells. The above paraffin blocks were entrusted to Shanghai Ming Code Technology Co., Ltd. for whole genome sequencing (WGS) and partial transcriptome sequencing (RNA-seq). The clinical classification of ESCC was based on the 8th edition of the TNM classification standard designated by the American Joint Committee on Cancer and the International Union Against Cancer. All specimens were collected with the consent of the patients and their families and signed informed consent. The clinical and pathological characteristics of patients with esophageal squamous cell carcinoma are shown in Table 1.

[0032] Table 1: Clinical and pathological characteristics of patients with esophageal squamous cell carcinoma

[0033]

[0034] 2. Cell lines: The human esophageal squamous cell carcinoma cell lines KYSE180, KYSE450, and KYSE150 used in this experiment were stored at low temperature. The main reagents and materials are shown in Table 2.

[0035] Table 2: Main reagents and materials

[0036]

[0037] II. Research and experimental methods

[0038] 1. Whole genome sequencing

[0039] First, whole genome sequencing of 528 samples was performed, which was completed by the company. According to the manufacturer's instructions, high-quality total DNA was extracted using the Maxwell 16 Tissue DNA Purification Kit (Promega). About 300 ng of high-quality DNA samples (od260 / 280 = 1.8~2.0) were cut to ~350 BP using a Covaris S220 ultrasonic instrument (Covaris). Sample purification beads (Illumina) were used to purify fragment DNA. The TruSeq Nano DNA Sample Preparation Kit (Illumina) was used to prepare adapter-ligated libraries, and the Illumina HiSeq system was used for double-end sequencing.

[0040] 2. Variant detection

[0041] CNV detection, CNV was detected by patchwork (http: / / patchwork.r-forge.r-project.org / ).

[0042] SV event detection, SV breakpoints were identified by SvABA (https: / / github.com / walaj / svaba) and Delly (https: / / github.com / dellytools / delly). The results of SvABA were filtered to remove non-significant SV events, and the results of Delly were manually checked. The CNV-related results were retained and combined with the results of SvABA as the final results.

[0043] 3. SV hotspot determination: SV hotspot determination in ESCC: To systematically study the TD hotspots in ESCC, the method proposed by Glodzik (https: / / www.nature.com / articles / ng.3771) was applied to different TD events and SV hotspots were identified. It uses the PCF algorithm to determine the genomic regions that show a rearrangement density much higher than that observed in adjacent genomic regions. This method was applied to all TD events to explore regions with a rearrangement density twice that of the whole-genome background density to identify candidate TD mutation hotspots.

[0044] 4. Determination of TD hotspots in enhancer regions: The detected TD mutation hotspots were compared with the H3K27ac chip data of esophageal cancer cells to confirm that the hotspots were located in the super-enhancer region upstream of the PTHLH gene. Data source: EC074 DNase sequence and H3K27ac chip sequence data were downloaded from the NIH Roadmap Epigenomics Mapping Consortium (http: / / egg2.wustl.edu / roadmap), and KYSE180 H3K27ac chip sequence data were downloaded from GEO.

[0045] 5. Identification of TD hotspots

[0046] A. Sequence acquisition and primer design: According to the TD event chromosomal breakage position detected by SV, the whole genome sequence was downloaded from the UCSC database, and primers were designed 1000 bp before and after the breakpoint. The product fragment needs to span the chromosomal breakage region, and the primer sequence is shown in Table 3.

[0047] Table 3: Primer sequence

[0048]

[0049] B. Whole genome DNA extraction, the specific method is as follows:

[0050] (1) First, the adherent culture cells are treated as cell suspension, centrifuged at 10000 rpm for 1 min, the supernatant is discarded, 200ul buffer GA is added, and oscillation is carried out until complete suspension;

[0051] (2) Add 20 ul Proteinase K solution and mix well;

[0052] (3) Add 200 ul buffer GB, mix well by overturning, and place at 70℃ for 10 min. The solution should be clear, and a brief centrifugation is performed to remove the water droplets on the inner wall of the tube cap.

[0053] (4) Add 200 ul anhydrous ethanol, mix well by oscillation for 15 s. At this time, a flocculent precipitate may appear, and a brief centrifugation is performed to remove the water droplets on the inner wall of the tube cap;

[0054] (5) The solution and flocculent precipitate obtained in the previous step are added to an adsorption column CB3, centrifuged at 12000 rpm for 30 s, and the waste liquid is discarded;

[0055] (6) Add 500 ul buffer GD, centrifuge at 12000 rpm for 30 s, and discard the waste liquid;

[0056] (7) Add 600 ul rinse liquid PW, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and repeat once;

[0057] (8) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, discard the waste liquid, and then place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material;

[0058] (9) Transfer the adsorption column CB3 into a clean centrifuge tube, and add 50-200 uI elution buffer TE to the middle part of the adsorption membrane, and place at room temperature for 2-5 min, centrifuge at 12000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0059] (10) Repeat step (9), take 1 ul of the collected product, and detect its purity and concentration.

[0060] C. Agarose gel electrophoresis

[0061] Preparation of agarose gel: dissolve 0.6 g agarose in 60 ml 1xTAE, heat in a microwave oven until completely dissolved; after cooling to room temperature, add 6 ul of nucleic acid dye, uniformly stir, and then pour the solution into the prepared mold, cool and reserve;

[0062] Take 15ul prepared DNA sample and DNA marker corresponding to add to the well of agarose gel, voltage 110V, run gel in TAE buffer for 30-40min; take the above agarose gel and take a photo by ultraviolet developing instrument, analyze the experimental results;

[0063] The pre-selected positive results are amplified by the system, separated by 1% agarose gel electrophoresis, and the agarose gel containing the target DNA fragment is cut with a clean knife and placed in a clean 1.5ml EP tube.

[0064] D, gel recovery:

[0065] (1) Weigh the total weight of the gel, and add 3 times the volume of XP2 Binding Buffer solution according to the principle of 100mg≈100ul, incubate in 55℃ metal bath for 10min until the gel is completely melted, and shake every 2-3min during the period to accelerate dissolution;

[0066] (2) Add the above mixture to the DNA collection tube, centrifuge at 10000rpm for 1min, and discard the waste liquid;

[0067] (3) Add 300ul XP2 Binding Buffer to the above collection tube, centrifuge at 10000rpm for 1min, and discard the waste liquid;

[0068] (4) Add 700ul SPW Buffer to the above collection tube, centrifuge at 13000rpm for 1min, discard the waste liquid; repeat once;

[0069] (5) Place the DNA collection tube in a new clean 2ml collection tube, dry at room temperature for 2min; then, add 20ul Elution Buffer, place at room temperature for 2min, centrifuge at 10000rpm for 1min;

[0070] (6) Repeat step (5), take 1ul of the collected product, and detect its purity and concentration.

[0071] E, sanger sequencing identification: take 30ul PCR gel recovery product, respectively take 10ul corresponding upstream and downstream primers to Beijing Huada Company for sequencing identification.

[0072] 6, enhancer vector construction

[0073] Sequence acquisition: According to the prediction of enhancer position on the chromosome by bioinformatics analysis, a total of 8 potential enhancer regions were analyzed, named e1-e8, specific primers were designed at both ends of the sequence, and the enhancer fragment was amplified with whole genome DNA as a template, and then sanger sequencing was performed (as above). The enhancer site and primer sequence are shown in Table 4.

[0074] Table 4: Enhancer site and primer sequence

[0075]

[0076] Enzyme digestion: The vector was double-digested with Kpn I and Xho I, and the double-digested reaction is shown in Table 5. The fragment was double-digested with Kpn I and Xho I, and the double-digested reaction of the fragment is shown in Table 6. Enzyme digestion reaction: 37°C, 2 hours.

[0077] Table 5: Double enzyme digestion reaction of vector enzyme digestion

[0078]

[0079] Table 6: Double enzyme digestion reaction of fragment enzyme digestion

[0080]

[0081] Enzyme digestion recovery: (1) The above enzyme digestion product was separated by 1% agarose gel electrophoresis;

[0082] (2) A clean knife was used to cut the agarose gel containing the target fragment as much as possible and placed in a clean 1.5ml EP tube, the gel weight was weighed, and according to the principle of 100mg≈100μl, 3 times the volume of XP2 Binding Buffer was added, 55℃ metal bath incubation for 10min to completely dissolve the gel, and during the period, shake once every 2-3min to accelerate the dissolution;

[0083] (3) The dissolved mixture of (2) was added to the DNA mini column, centrifuged at 10000rpm for 1min, and the waste liquid was discarded;

[0084] (4) 300μl XP2 Binding Buffer was added to the DNA mini column, centrifuged at 10000rpm for 1min, and the waste liquid was discarded;

[0085] (5) 700μl SPW Buffer was added, centrifuged at 13000rpm for 1min, the waste liquid was discarded, and the operation was repeated once;

[0086] (6) Put the DNA mini column into a new clean 2ml collection tube, dry at room temperature for 2min;

[0087] (7) Add 20μl Elution Buffer to the DNA mini column, place at room temperature for 2min, centrifuge at 10000rpm for 1min; take 1μl of the collected product for purity and concentration detection.

[0088] Ligation reaction: mix the vector and the inserted DNA fragment according to 1:1 to 1:10 molar ratio to prepare 10μl of DNA mixture; add equal volume of Solution I (total volume not more than 20μl) to the above mixture, mix well and place in 16℃ water bath for 2h.

[0089] Transformation: (1) take 100μl of DH-5α competent bacteria and transfer into a clean 1.5ml EP tube, add the mixture of ligation product and solution III (9:1), mix gently and place on ice for 30min;

[0090] (2) after heat shock at 42℃ for 90s, quickly place the reaction tube on ice for 2-3min;

[0091] (3) add 500μl of LB liquid medium without antibiotic to the above reaction tube, shake culture at 200rpm and 37℃ for 1h;

[0092] (4) add appropriate amount of bacterial liquid in (3) to LB solid medium containing antibiotic, evenly spread with a sterile glass rod, after the bacterial liquid on the plate surface is dried, culture at 37℃ overnight.

[0093] Colony PCR identification: sterile inoculation loop to pick up a single morphologically full colonies on the plate, draw a line and number on the plate containing antibiotic, and incubate at 37℃ for 6-8h; pick up the bacterial body of each clone, and perform colony PCR reaction, the reaction system is shown in Table 7. The PCR product is identified by 1% agarose gel electrophoresis, and the clone consistent with the expected result is selected for expansion culture; send to the company for sequencing identification.

[0094] The nucleotide sequence of enhancer Enhancer e3 (hg19 chr12: chr12:28176001-28177500) is shown as SEQ ID NO: 1; the nucleotide sequence of enhancer Enhancer e5 (hg19 chr12: chr12:28185600-28186900) is shown as SEQ ID NO: 2; and the nucleotide sequence of enhancer Enhancer e8 (hg19 chr12: chr12:28284501-28285500) is shown as SEQ ID NO: 3.

[0095] Table 7: PCR reaction system

[0096]

[0097] Plasmid extraction and preservation: according to the sequencing results, 500ul of correct bacterial liquid was taken into a sterile cryotube, 500ul of 40% glycerol was added, and it was mixed and stored at -80℃ after mixing; the remaining bacterial culture solution was transferred to a 15ml EP tube, centrifuged at 8000 rpm for 10min, the supernatant was discarded, and the bacterial precipitate was collected;

[0098] First, resuspend the bacterial precipitate with 500ul Buffer P1, then add 500ul Buffer P2, mix well for 4-6 times, then add 500ul Buffer P4, mix well for 4-6 times, stand for 10min, centrifuge at 12000 rpm for 10min; gently take out the EP tube from the centrifuge, and carefully transfer the supernatant to the CS filter column (note not to suck the white precipitate at the bottom), incubate at room temperature for 2min, centrifuge at 12000 rpm for 2min, and collect the filtrate in a clean 2ml centrifuge tube;

[0099] Add 1 / 3 volume of isopropanol to the filtrate, mix well and transfer to the pre-activated CP4 tube, centrifuge at 12000 rpm for 1min, discard the waste liquid; repeat until all the liquid is centrifuged; wash the CP4 column with 500ul deproteinization PD, centrifuge at 12000 rpm for 1min, discard the waste liquid; wash the CP4 column with 600ul Buffer PW, centrifuge at 12000 rpm for 1min, discard the waste liquid; empty the CP4 column by centrifuging at 12000 rpm for 2min, discard the lower tube, and place the CP4 column in a new clean 2ml collection tube, dry at room temperature for 2min;

[0100] Add 200ul of preheated Buffer TB to the centrifugal column, incubate at room temperature for 2-3min, centrifuge at 12000 rpm for 1min, collect the eluate, and repeat once; take 2ul for purity and concentration identification, and store at -20℃.

[0101] 7. Cell culture

[0102] Cell recovery: Take the cells to be recovered from liquid nitrogen, immediately placed in a 37°C water bath to make it quickly and completely melted, 800 rpm centrifugation for 5 min, discard the supernatant; add 1 ml complete medium resuspend the cell pellet and transfer to a 6 cm dish, placed in a 37°C, 5% CO2 incubator for culture.

[0103] Cell passage: When the cell confluence reaches more than 80%, the cell passage is needed. Discard the original culture medium, add appropriate amount of balanced to room temperature 1x PBS buffer to wash the cells twice, add appropriate amount of 0.25% trypsin, 37°C, 5% CO2 incubator for 2-3 min, observe under microscope that most cells are round and float, which means digestion is complete, immediately add an equal amount of complete medium to terminate digestion, transfer the cell suspension to a centrifuge tube, centrifuge at 800 rpm for 5 min, discard the supernatant, add appropriate amount of complete medium to resuspend the cell pellet and distribute to 2-3 culture dishes.

[0104] Cell cryopreservation: Use the conventional method to digest and collect the logarithmic growth phase cells, discard the supernatant, add appropriate amount of cell cryopreservation solution (DMSO: FBS = 1:9) to resuspend the cell pellet, distribute to sterile cryopreservation tubes, according to the principle of gradient cryopreservation, sequentially: 4°C for 30 min--20°C for 2 h--80°C overnight-liquid nitrogen long-term preservation.

[0105] 8. Dual luciferase reporter assay

[0106] Cell transfection:

[0107] (1) KYSE150 and KYSE450 cells were inoculated into 24-well plates at 5x10 4 cells per well, and the next day when the cell confluence reached 60%-70%, plasmid transfection was performed, with three replicates for each group;

[0108] (2) Add 1.5 ug of recombinant vector and empty vector to 150 ul of Opti-MEM medium, and incubate at room temperature for 5 min;

[0109] (3) Add 4.8 ul of EL to 150 ul of Opti-MEM medium, and incubate at room temperature for 5 min;

[0110] (4) Add the mixture in tube (3) dropwise to tube (2), mix gently, and incubate at room temperature for 20 min;

[0111] (5) Discard the old cell culture fluid, dropwise add the mixed solution in (4), complete medium to 500ul, 37℃, 5%CO2 culture, 6h after changing the liquid, continue to culture to 48h, during close observation of the state of the transfected cells.

[0112] Dual luciferase reporter experiment: after 48h of transfection, remove the cell culture fluid, rinse twice with 1xPBS, add 100ul of cell lysis solution, fully lyse at room temperature for 10min, scrape the cells into 1.5ml EP tubes, centrifuge at 4℃, 12000rpm for 10min, and reserve the supernatant; add 100ul of luciferase reaction reagent balanced to room temperature to the 1.5ml EP tube, then carefully add 20ul of cell lysis solution, mix gently, centrifuge briefly, and detect the activity of firefly luciferase reporter gene in the chemiluminescence instrument; remove the reaction tube, add 100ul of luciferase reaction reagent II balanced to room temperature, mix gently, centrifuge briefly, and detect the activity of sea anemone luciferase reporter gene on the chemiluminescence instrument; compare the ratio of the activity of the target luciferase reporter gene to the activity of the internal reference luciferase reporter gene in each group, and draw a chart.

[0113] 9、Enhancer knockout experiment: construct MCS-EF1a-Cas9-FLAG-P2A-puro plasmid targeting e3 and e5 enhancers. The virus MOI value is preset as 1, 5, 10, 20, 50, and 100. Transfect the experimental group and control group viruses into cells, and after 2 weeks of puro screening, expand the culture, extract genomic DNA to detect the knockout efficiency of the SE region, and extract RNA to detect the expression of PTHLH gene, so as to verify the influence of knocking out the upstream SE region of PTHLH on the expression of PTHLH.

[0114] 10、PTHLH-siRNA interference and efficiency identification:

[0115] A, PTHLH-siRNA transfection: inoculate cells: inoculate KYSE150 and KYSE180 cells into 6-well plates, 1.2x10 5 cells per well, and transfect when the cell density reaches 30%-50%; dilute siRNA: dilute 1.10ul of 20uM siRNA stock solution with 120ul of riboFECT TM CPBuffer, mix gently; prepare the mixed solution: add 12ul of riboFECT TM CPreagent, mix gently, incubate at room temperature for 15min, and prepare the transfection complex; add the transfection complex to the appropriate amount of double-antibody-free complete culture medium, mix gently. Place the culture plate in a 37℃ CO2 incubator for 24-96h.

[0116] B. Transfection efficiency identification:

[0117] RNA extraction:

[0118] (1) Add 600ul Trizol into the fresh collected cell precipitate, and stand at room temperature for 10 min;

[0119] (2) Add 120ul chloroform into (1), mix well by inverting 4-6 times, stand at room temperature for 5 min, centrifuge at 12000 rpm at 4℃ for 15 min;

[0120] (3) Carefully transfer the supernatant to a new clean 1.5 ml EP tube, add equal amount of isopropanol, stand at -20℃ for 30 min, centrifuge at 12000 rpm at 4℃ for 10 min;

[0121] (4) Discard the supernatant, observe the position of the precipitate at the bottom of the tube, add 1 ml 70% ethanol to wash the precipitate, centrifuge at 12000 rpm at 4℃ for 5 min, discard the washing solution, and dry the precipitate at room temperature for 3 min;

[0122] (5) Add 30-50ul RNase free water to dissolve the precipitate;

[0123] (6) Take 2ul for purity and concentration identification, and store at -20℃.

[0124] cDNA template preparation: The cDNA reaction system is shown in Table 8. The cDNA reaction conditions are as follows: 37℃, 15 min; 85℃, 5 s; -20℃ storage.

[0125] Table 8: cDNA reaction system

[0126]

[0127] Fluorescent quantitative PCR detection: The fluorescent quantitative PCR reaction system is prepared as shown in Table 9. The fluorescent quantitative reaction conditions are as follows: 95℃, 10 min; 95℃, 15 s; 60℃, 1 min, for a total of 40 PCR cycles. The results are determined as follows: the housekeeping gene of the control group is used as an internal reference, 3 replicate wells are set for each well, and the △△ Ct method is used to calculate the relative expression amount.

[0128] Table 9: Fluorescent quantitative PCR reaction system

[0129]

[0130] The PTHLH-siRNA and primer sequences are shown in Table 10.

[0131] Table 10: PTHLH-siRNA and primer sequences

[0132]

[0133] 11. Cell line function experiment

[0134] CCK8 experiment: Inoculation of cells: KYSE150 and KYSE180 blank group and experimental group cells were inoculated into 96-well plates at 5x10 3 cells per well, and 4 time points were set, with 7 replicate wells for each time point. The number of living cells cultured for 24 h, 48 h, 72 h and 96 h was detected. The original culture medium was discarded, 10% CCK solution was added, and the cells were further cultured at 37°C and 5% CO2 for more than 30 min. The absorbance value at an excitation wavelength of 450 nm was detected by an enzyme-labeled instrument. The absorbance values detected for 4 consecutive days were used as the vertical coordinate, and the time points were used as the horizontal coordinate to draw a cell growth curve.

[0135] Hard cloning experiment: Inoculation of cells: KYSE150 and KYSE180 blank group and experimental group cells were inoculated into 6-well plates at 800 cells per well, and cultured at 37°C and 5% CO2 for 7-12 days. Cell clones were observed under a microscope, and a total of more than 50 cells were counted as one clone. The white clones were visible to the naked eye and the culture was stopped. The old cell culture medium was discarded, and the cells were gently washed twice with appropriate amount of 1x PBS buffer. 1 ml of 4% paraformaldehyde was added for room temperature fixation for 15 min, and the cells were washed 4 times with 1x PBS buffer. Appropriate amount of crystal violet was added for room temperature staining for 30 min, and the cells were washed 4 times with 1x PBS buffer. The number of clones was counted under a microscope.

[0136] Wound healing experiment: Inoculation of cells: First, evenly draw horizontal lines on the back of a 6-well plate at an interval of 1 cm using a marker pen. Then, KYSE150 and KYSE180 blank group and experimental group cells were inoculated into the 6-well plate at 9.5x10 5 cells per well, and cultured at 37°C and 5% CO2 overnight. The next day, when the cell density reached 90%, a wound was drawn perpendicular to the horizontal line on the back using a 10ul gun tip, taking care not to tilt. The cells floating up were washed away with PBS, and serum-free medium was added. The culture was placed in a 37°C, 5% CO2 incubator, and samples were taken at 0, 24, and 48 hours for photography. The area was analyzed using image J software, and the migration rate was calculated.

[0137] Invasion and migration experiment: For the invasion experiment, BD matrigel glue was diluted with serum-free basal medium at a ratio of 1:6 4h in advance, 100ul was added to each well to avoid the generation of bubbles, and the culture was incubated at 37°C for 4h to make it solidify. Inoculation of cells: KYSE150 and KYSE180 blank group and experimental group cells were inoculated into the Transwell chamber at 1.2x105 The cells were cultured for 24 h and 48 h, respectively. The cells in the inner side of the chamber that did not undergo invasion and migration were wiped off with a clean and dry cotton swab. The chamber was fixed in 4% paraformaldehyde at room temperature for 15 min and washed 4 times with 1x PBS buffer. The chamber was stained with crystal violet at room temperature for 20 min and washed 4 times with 1x PBS buffer. After drying, the chamber was placed on a clean glass slide and observed under a microscope. The number of invasive cells was counted in 5 randomly selected fields.

[0138] 12. Statistical analysis: t-test was used for comparison of means of measurement data; chi-square test was used for count data; single factor ANOVA variance analysis was used for comparison of means among multiple samples; spearman regression analysis was used for correlation between two factors; statistical software R 4.0 and SPSS 19.0 were used.

[0139] III. Experimental results

[0140] 1. TD drives and amplifies the upstream super-enhancer of PTHLH gene in ESCC

[0141] Based on the whole-genome sequencing results of 528 cases of ESCC, the statistical results showed that SV events frequently affected driver genes. These affected regions not only contained these genes but also involved their regulatory regions, including a new potential driver gene PTHLH.

[0142] The statistical results showed that a total of 85 samples detected significant amplification of Focal CNV in the upstream regulatory region of the PTHLH gene (85 / 522, 16.28%, p<2.2e-16). Figure 1 To verify the accuracy of the TDs composed of PTHLH, Sanger PCR verification was performed on 11 TDs, and the experimental results confirmed that these TDs were real SV events. Figure 2 After including the enhancer results of ESCC cell lines, DNase-seq and H3K27ac chromatin data showed that there was a significant peak aggregation at this position. At the same time, the region where this Focal CNV was located did not exceed the boundary of TAD. Figure 1 Different sequences of the peak region were selected for dual luciferase reporter experiments in KYSE450 cell lines, and the results showed that there were multiple enhancer-active regions. Figure 3 A, B).

[0143] 2、Super-enhancer region amplification promotes PTHLH gene expression increase: Using Cas9 technology to knock out the corresponding sequence of the super-enhancer, detect the expression change of PTHLH gene in cells, the results show that after knocking out part of the super-enhancer region, the expression of PTHLH gene is significantly reduced (Fig. 2A). Figure 3 C). Based on the transcriptome sequencing of some of the above samples, the results show that the RNA expression of samples with SE region amplification upstream of PTHLH gene is significantly higher than that of other samples (Fig. 2B). Figure 4 A). The results prove that the upstream super-enhancer driven by TD event and amplified in ESCC can significantly promote the expression of PTHLH gene.

[0144] 3、PTHLH gene is highly expressed in ESCC: Based on the results of transcriptome and protein expression statistics, the RNA and protein expression of PTHLH in ESCC is significantly higher than that in the adjacent control tissue (P = 2.54E-14 & P = 0.00029, Figure 4 B, C).

[0145] 4、PTHLH is related to prognosis and metastasis in ESCC: Through clinical correlation analysis, in 528 ESCC patients, the proportion of metastasis is higher (P = 0.023) and the prognosis is worse (P = 0.073) in patients with copy number amplification than in patients without amplification (Fig. 3A). In addition, in 119 patients, the proportion of metastasis is higher (P = 0.013) and the prognosis is worse (P = 0.058) in patients with up-regulated PTHLH protein expression than in patients without up-regulation (Fig. 3B). The statistical results preliminarily show that PTHL gene has a promoting effect on ESCC and is a potential oncogene. Figure 5 Figure 5 B). The statistical results preliminarily show that PTHL gene has a promoting effect on ESCC and is a potential oncogene.

[0146] 5、PTHLH as a cancer gene affects the proliferation and metastasis of ESCC cells: In order to verify the cancer gene effect of PTHLH, we carried out cell function experiments in two ESCC cell lines KYSE180 and KYSE450 respectively. After knocking out PTHLH gene, cell proliferation experiment, scratch experiment and invasion and migration experiment were carried out, the results showed that after knocking out PTHLH gene, the proliferation and metastasis ability of cells were significantly reduced (Fig. 4, Figure 6 , 7), which confirmed our analysis conclusion.

[0147] In summary, these findings suggest that PTHLH is a potential driver target, mainly driven by the amplification of the upstream super-enhancer in ESCC to affect its expression.

[0148] ​It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the above embodiments of the present application have been described in detail, those skilled in the art should understand that the technical solutions recorded in the above embodiments can be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application. SEQUENCE LISTING <110> shenzhen beijing university-hong kong science and technology university medical center <120> application of gene PTHLH as a target point in auxiliary diagnosis, prognosis and treatment of esophageal squamous cell carcinoma <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1500 <212> DNA <213> Artificial Sequence <400> 1 tctgaaagct tacaatcgtg gcagaaggca aagctggagc ttgcacgtca cctggggaaa 60 gcaggagcaa gagagtggga ggggaggtgc cacacacttt taaatgacca gatgtcatga 120 gaacacactc actatcatga ggacagcacc aaggggatgg cgttaaatca ttcatgagaa 180 atctgcccca tgatccaatc acatcccacc aggccccacc tccaacactg gggattacat 240 ttcaatatga gatttgggtg gggacacaca tccaaactct atcactgtga ctcagggttt 300 ctcacaaggc ttcaactaag atattcaaca gggctgcagt catcttagag ttcaactggg 360 gaggatacac ttccaagctc gctcaagtga ctgttggcag gcatcaggcc cttgctgctg 420 ttgaccggag acatcagttc cttgccacat gggcccctct gtagggcagc tcacaacagg 480 gcagctcaca acatggcagc tcacttccct cagaacaagc cagtgggaga gtgagagggg 540 aggccaaagc cagaagccac agattttttt gccctctaat gtcggaagtg acagctcatc 600 attttcgact tattctgttc attagaagtg agtcactagg tccagctcat gctcaaaacc 660 aggaaattac acaaaggcat gaataccagg aggtggagat cactgtgggg ggtaggggag 720 gtgcatttta gaggccacct gggaaaaatg gggctgtagc ttatttacta atgtttgtgt 780 ttttatattt tcaatgtttg acatagtatc tggctcaaca caatcttacc cctatggcta 840 agattttctt cgttctggga aatatgagag attcctcatt gtcagttgcc cacatagcat 900 tgatttcatt gctctatttt aagggaacag caattaccaa ggcaagacac catgaaaggt 960 ggaagaatgt agcctgcagc ctgtcccacc gtttgggaca ggtgggatgg actgccggat 1020 agcgtgtagg aagaagatta aaacaaatgg gggaaattta cacacatcat ttatttatct 1080 aaaatgttct ccctgtcata gtctattccc tctttaaact gtggaaagaa aagccctagc 1140 ataaacttgg tgtacctttt aaaaattatt acctggtact tcatttaaaa gaaaaggaga 1200 aattataatt aaccagttaa gactcacctt ttctgacata gcatgtggtt gtatacatct 1260 ggttgtaacc atggttcaag ttgatcaacc acctaccagt tggctaaata aattcctgat 1320 atgctaggca tctgggatga acttccctca gacaagcctt tagctcatgg ctcataagaa 1380 tgaatcttgt tttgtatgat gtcattgagt gtggagatat gctgatggaa agatggtgct 1440 tagcaaccaa ctacttattt tctttagtct tcctacaggg taaccacgaa gtctttctgc 1500 <210> 2 <211> 1301 <212> DNA <213> Artificial Sequence <400> 2 tctgaaaggc tagtacactc tttgattgca aggtaagtaa aaacctttta gtagaaatca 60 ttctggatat ttcacaccac tttatgctac aggatcactg atctgagcat aaattcacat 120 ttttaacttg ataataaata tagaagataa acaagatgag tcagaaaaga atacttgcga 180 atacctggtt agacctggga actggttggt taaaaagatg aatccctaat gtcaaatgac 240 tttcttgtga ttcacttcat ttctcgtgga agctaaatga aaagcaaaaa tgttaattga 300 tggttgtggt aagtcaggct ccagttgact aaggttttct tttgtctaaa cccatctcca 360 gggtgaataa atagaactac tccgtgaaag aacaaaattg atgtggtctg ttacctctgg 420 atcatctcaa ggaacaatac tttcaaagcc tagcaacttg caaacttcct gtctgagaag 480 gattggtggt agggggatgc ttgggaacat aacacccctg ccccacacac cccacaccag 540 ctgcttagaa ggcaggaagc aggatccagg cctccaggaa atgtgcttta tgggatcttg 600 tccacacata tttgggtctt cagcaacctg tcctaacctg ctttattta gtgactgtta 660 ctaacttttt aagctctgaa tcagcaaact ctctccccca ctttttttt cccctgagga 720 cattcaaaga tcatccagcc cactttagtt aaagtagaaa tagtggcatg gtggctgtgg 780 gtctggtggt gtcacttcaa agcaaatgag atacccttga tgattctggc aaggtgtggt 840 ctcccactgt cggtctctcc cckatatg atgaaggtac tttttaggtg gtaaaggaat 900 gtccttgccc agtggtagtt aggtgtaaat actctacatt ttatattttt tcttttcatg 960 ttgaatatca aacgtggtat aaatgatgaa accgatcact gcattattcc caggcctttt 1020 acttagtgtg tctctgctgt tagcatttgc atggatctgt gtcatcattc tgattcatcc 1080 tgctgaataa tctatcattt ctgctccctt ttccccatcc tccaacatcc tactcagggc 1140 tgtttcaggt gattctgaga cccattgtat cttctacaga tgaagcactt tcctgaaatt 1200 cctggatata gtcttctgat cagtggattt aagcaatgta catgaagttt atatgcggat 1260 ttaagtctat ggatttaaat gatggagagc atcctttttt t 1301 <210> 3 <211> 1000 <212> DNA <213> Artificial Sequence <400> 3 tattcagagg tgggtggaga aacagaacaa gagactggaa gaaaaaaaac ctgtagatca 60 aaagatcgag atgaagactt tgccacttac ttgcttcatg acattgggca gaatacagtg 120 atgcacttaa cctctgtgaa ctttaagatt ccttatctac aaaataggtt aatagtacta 180 acgactccag tggatttatg tgcaagtcac ctcaattctg taaggtaagg agtatcactc 240 cctattttat agataaggaa actatggtat aagatttttt ttctgtttgt tataatgatt 300 cccataaaac ctgtaacagt tctctacatg caacaaaaat aagtacttgt tgattggtca 360 ctttatgtac ataggtatct taatggaaga gactatttat caaatagaga atctcttatc 420 cagccatgaa gtaggtgcac actgtaagat gccttcctgg ggtggttggc acagatgatc 480 tctcaatgtt cctttcagtt atataattct gggatgtcaa attgaaactg aacaaagaac 540 ataactggca aaaatgaaat tactgctttt ctggagacag tgctatttgt ctgaaaatgg 600 taaaatgtag atgggaagga ggaaataaca ggtctgtgat tcatgatact aaaagctaag 660 aaaaggtggg ttttccttgt ggctataaaa caaacaaaaa agcatataaa cccagtgttt 720 aaatgttaat ttgctacagt acatcaaatg tttatagcaa gaatttgatg gaagcatcct 780 attgaaaaaa acaaattctt tcctatgtca atgtgcttat aaagaggata cccaactgtc 840 tccagcaatg catatttatg tctctgcaga gaataacctg accttatttg ctgcagtgac 900 catgaaacag tgcttccata atggtaatat gaaatatttc agaagaacag aaaaccttgt 960 ggtttcttag ataaaagaaa atactaccct atcttgcatt 1000

Claims

1. Use of a reagent for detecting expression level of a target gene PTHLH in preparation of a reagent for diagnosing esophageal squamous cell carcinoma.

2. Use of a reagent for detecting expression level of a target gene PTHLH in preparation of a reagent for evaluating prognosis of esophageal squamous cell carcinoma.

3. Use of a reagent for detecting a super-enhancer in the preparation of a reagent for diagnosing esophageal squamous cell carcinoma, characterized in that: The super-enhancer is a tandem repeat accumulated super-enhancer, which is an upstream regulatory region of PTHLH, and has a nucleotide sequence of: enhancer Enhancer e3, located at hg19 chr12: chr12: 28176001-28177500, and a nucleotide sequence as shown in SEQ ID NO: 1, or enhancer Enhancer e5, located at hg19 chr12: chr12: 28185600-28186900, and a nucleotide sequence as shown in SEQ ID NO:

2.

4. Use according to claim 1 or 2, characterized in that: The PTHLH gene is specifically up-regulated in cancer tissues of esophageal squamous cell carcinoma patients, and the protein expressed by the PTHLH gene is specifically up-regulated in cancer tissues of esophageal squamous cell carcinoma patients.

5. Use according to claim 3, characterized in that: Specific expression level of the tandem repeat accumulated super-enhancer is significantly up-regulated in cancer tissues of esophageal squamous cell carcinoma patients, which significantly up-regulates the expression of the PTHLH gene.

6. Use of an agent that inhibits expression of the gene PTHLH in the manufacture of a medicament for the treatment of esophageal squamous cell carcinoma, characterized in that: The reagent is: siRNA1: GGCGACGATTCTTCCTTCA; siRNA2: CGTCCGATTTGGGTCTGAT.

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