CN115044673A - Application of reagent for detecting HN1L expression in preparation of esophageal squamous carcinoma diagnostic kit - Google Patents

Application of reagent for detecting HN1L expression in preparation of esophageal squamous carcinoma diagnostic kit Download PDF

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CN115044673A
CN115044673A CN202210550398.0A CN202210550398A CN115044673A CN 115044673 A CN115044673 A CN 115044673A CN 202210550398 A CN202210550398 A CN 202210550398A CN 115044673 A CN115044673 A CN 115044673A
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hn1l
esophageal squamous
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李雷
曾婷婷
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Sun Yat Sen Memorial Hospital Sun Yat Sen University
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Abstract

The invention discloses the application of a reagent for detecting HN1L expression in preparing an esophageal squamous carcinoma diagnostic kit, and through in vitro and in vivo functional experiments, the knocking-down HN1L is found to inhibit the metastasis and proliferation of esophageal squamous carcinoma cells and improve the sensitivity of tumor cells to chemotherapeutic drugs, so that the reagent can be used as a specific marker gene for diagnosing esophageal squamous carcinoma, and the diagnosis of the esophageal squamous carcinoma is more accurate and rapid; the target can be used for preparing a medicine target for treating esophageal squamous cell carcinoma, and a new effective treatment scheme is provided, namely the target HN1L is combined with a chemotherapeutic medicine for treating esophageal squamous cell carcinoma, so that a new way is provided for treating esophageal squamous cell carcinoma.

Description

Application of reagent for detecting HN1L expression in preparation of esophageal squamous carcinoma diagnostic kit
Technical Field
The invention relates to the technical field of biological medicines, in particular to application of a reagent for detecting HN1L expression in preparation of an esophageal squamous cell carcinoma diagnostic kit.
Background
Esophageal cancer is a common malignancy of the digestive system. The survival rate of patients with esophageal squamous carcinoma is low, mainly because of late clinical symptoms and lack of early diagnostic markers. Therefore, there is an urgent need for new highly sensitive, cost effective biomarkers and therapeutic targets for the diagnosis and treatment of esophageal squamous carcinoma.
The HN1L gene, also known as JPT2, C16orf34 or L11, encodes a protein of 190 amino acids (NCBI Reference Sequence: NP-653171.1). The protein is mainly localized in nucleus, cytoplasm and cell membrane, and is specifically expressed in liver, kidney, prostate, testis, uterus and other organ tissues. Gene HN1L was first identified in 2000 from a mouse fertilized egg cDNA library. At present, HN1L is found in the prior art to play a cancer promotion role in the development and development of tumors: HN1L promotes invasion and metastasis of esophageal-gastric junction adenocarcinoma (PMID:33471419), HN1L promotes migration and invasion of breast cancer by up-regulating expression of HMGB1 (PMID:33191617), HN 1L-mediated transcription axis AP-2 gamma/METTL 13/TCF3-ZEB1 drives tumor growth and metastasis of hepatocellular carcinoma (PMID:30778199), HN1L promotes triple-negative breast cancer stem cells by LEPR-STAT3 pathway (PMID:29249663), HN1L overexpression promotes malignant proliferation of non-small cell lung cancer cells (PMID:29053395), and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides an application of a reagent for detecting HN1L expression in preparing an esophageal squamous cell carcinoma diagnostic kit, and the knock-down of HN1L is found through in vitro and in vivo functional experiments to inhibit the metastasis and proliferation of esophageal squamous cell carcinoma cells and improve the sensitivity of tumor cells to docetaxel or cisplatin. Therefore, the HN1L gene and the expression product thereof can be used as a diagnostic marker and a drug target of esophageal squamous carcinoma, and the targeted HN1L combined with chemotherapeutic drugs is a new effective treatment scheme of esophageal squamous carcinoma.
The first purpose of the invention is to provide the application of the reagent for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in preparing an esophageal squamous carcinoma diagnostic kit.
The second purpose of the invention is to provide application of a reagent for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in preparing an esophageal squamous cell carcinoma prognosis prediction kit.
The third purpose of the invention is to provide the application of the reagent for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in the preparation of the sensitivity detection kit for the chemotherapy of patients with esophageal squamous cell carcinoma.
The fourth purpose of the invention is to provide the application of the inhibitor of HN1L gene expression, the inhibitor of HN1L mRNA expression or/and the inhibitor of HN1L protein expression in preparing the esophageal squamous carcinoma medicine.
The fifth purpose of the invention is to provide the application of the inhibitor of HN1L gene expression, the inhibitor of HN1L mRNA expression or/and the inhibitor of HN1L protein expression in the preparation of the synergist of the esophageal squamous carcinoma chemotherapeutic drug.
The sixth purpose of the invention is to provide an esophageal squamous carcinoma medicament.
In order to achieve the purpose, the invention is realized by the following scheme:
the application of the reagent for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in preparing an esophageal squamous carcinoma diagnostic kit, wherein the expression level of HN1L gene or/and HN1L protein of an esophageal squamous carcinoma patient is higher than that of a patient without esophageal squamous carcinoma.
Preferably, the esophageal squamous carcinoma diagnostic kit is a kit for diagnosing tumor tissue invasion, and a patient with high expression of HN1L gene, HN1L mRNA or/and HN1L protein has relatively low expression, and the tumor tissue invasion is severe.
Preferably, the esophageal squamous carcinoma diagnostic kit is a kit for diagnosing lymph node metastasis, and the HN1L gene, HN1L mRNA or/and HN1L protein are highly expressed in a patient, and relatively lowly expressed in a patient, the lymph node metastasis is severe.
Preferably, the esophageal squamous carcinoma diagnostic kit is a kit for diagnosing staging conditions, and the staging is late for patients with high expression of HN1L gene, HN1L mRNA or/and HN1L protein and relatively low expression patients.
The invention also claims the application of reagents for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in preparing an esophageal squamous carcinoma prognosis prediction kit, wherein patients with high HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression are relatively low-expression patients, and the prognosis is poor.
The invention also claims the application of a reagent for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in preparing a sensitivity detection kit for chemotherapy of patients with esophageal squamous carcinoma, wherein the sensitivity of chemotherapy is poor for patients with high HN1L gene expression, high HN1L mRNA expression or/and high HN1L protein expression and relatively low HN1L gene expression.
Preferably, the drug used in chemotherapy is docetaxel or cisplatin.
The invention also claims the application of the inhibitor of HN1L gene expression, the inhibitor of HN1L mRNA expression or/and the inhibitor of HN1L protein expression in preparing the esophageal squamous carcinoma medicine.
The invention also claims the application of the inhibitor of HN1L gene expression, the inhibitor of HN1L mRNA expression or/and the inhibitor of HN1L protein expression in the preparation of a synergist of an esophageal squamous carcinoma chemotherapeutic drug.
Preferably, the chemotherapeutic agent is docetaxel or cisplatin.
The invention also claims an esophageal squamous carcinoma drug, which contains an inhibitor of HN1L gene expression, an inhibitor of HN1L mRNA expression or/and an inhibitor of HN1L protein expression.
Preferably, the composition also contains a chemotherapeutic drug, wherein the chemotherapeutic drug is docetaxel or cisplatin.
Compared with the prior art, the invention has the following beneficial effects:
according to in vitro and in vivo functional experiments, the knocking-down HN1L is found to inhibit the metastasis and proliferation of esophageal squamous carcinoma cells and improve the sensitivity of tumor cells to chemotherapeutic drugs, so that the tumor cells can be used as specific marker genes for diagnosing esophageal squamous carcinoma, and the diagnosis of esophageal squamous carcinoma is more accurate and rapid; the target can be used for preparing a medicine target for treating esophageal squamous cell carcinoma, and a new effective treatment scheme is provided, namely the target HN1L is combined with a chemotherapeutic medicine for treating esophageal squamous cell carcinoma, so that a new way is provided for treating esophageal squamous cell carcinoma.
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FIG. 1 is a diagram showing the analysis of the expression level of HN1L gene in esophageal squamous carcinoma tissue by using esophageal squamous carcinoma tissue chip; (A) in The TCGA (The Cancer Genome Atlas) database, HN1L at mRNA levels was expressed higher in esophageal squamous carcinoma (ESCC) than in esophageal Adenocarcinoma (ADC) and normal esophageal epithelial tissue; (B) immunohistochemical staining analysis of the expression level of HN1L in esophageal squamous carcinoma and adjacent carcinoma paracsophageal epithelial cells; (C) immunohistochemical staining analysis of the expression level of HN1L in esophageal squamous carcinoma; (D) HN1L was scored for staining in normal tissues and esophageal squamous carcinoma tissues.
FIG. 2 is a correlation analysis of the expression level of HN1L in esophageal squamous carcinoma tissues and clinical pathological features; (A) high expression of HN1L was associated with tumor tissue invasion, lymph node metastasis and later staging; (B) high expression of HN1L in ESCC tissues suggests poor prognosis in patients with esophageal squamous cell disease.
FIG. 3 shows the results of cell metastasis experiments performed after knockdown of HN1L in esophageal squamous carcinoma cells; (A) knocking down HN1L by using a lentivirus transfection technology, and detecting the expression level of HN1L on esophageal squamous carcinoma cells KYSE150 by using Western blot; (B) in vitro Transwell experiments showed that knock-down of HN1L decreased tumor cell migration ability. (C) The nude mouse tail vein lung metastasis experiment shows that the knock-down of HN1L significantly inhibits the metastatic ability of tumor cells.
FIG. 4 shows the results of cell growth experiments performed after knockdown of HN1L in esophageal squamous carcinoma cells; (A) in vitro CCK8 cell proliferation experimental analysis shows that knocking down HN1L inhibits tumor cell proliferation; (B) the nude mice subcutaneous transplantation tumor experiment shows that the formed transplantation tumor weight is smaller after knocking down HN1L (3 mice in each group); (C) immunohistochemical staining analysis showed a lower proportion of proliferating cells (Ki67 positive) after knockdown of HN 1L.
FIG. 5 shows the results of sensitivity of cells to docetaxel following knockdown of HN1L in esophageal squamous carcinoma cells; (A) in vitro CCK8 cell proliferation experimental analysis shows that knocking down HN1L improves the sensitivity of tumor cells to docetaxel drugs; (B) nude mice subcutaneous transplantation tumor and drug experiments show that silencing HN1L can reverse the resistance of tumor cells to docetaxel drugs.
FIG. 6 shows the results of cisplatin sensitivity of cells following knockdown of HN1L in esophageal squamous carcinoma cells: nude mice were implanted subcutaneously and drug experiments showed that silencing HN1L reversed tumor cell resistance to cisplatin drugs (3 mice per group).
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 HN1L Gene is highly expressed in esophageal squamous carcinoma tissue
First, experiment method
The mRNA expression level of HN1L gene in normal esophageal tissue, esophageal squamous carcinoma and esophageal adenocarcinoma was obtained by using an online analysis tool in UALCAN (http:// UALCAN. path. uab. edu/analysis. html). The data source for this analysis tool is the TCGA database.
Second, experimental results
As a result, as shown in fig. 1A, HN1L gene was expressed higher in esophageal squamous cell carcinoma (n-95) and esophageal adenocarcinoma (n-89) than in normal esophageal tissue (n-11, P < 0.001). Meanwhile, the expression level of HN1L gene in esophageal squamous carcinoma is higher than that in esophageal adenocarcinoma tissue (P < 0.001).
Example 2 HN1L protein is highly expressed in esophageal squamous carcinoma tissue
First, experiment method
Immunohistochemical staining was performed on the esophageal squamous cell carcinoma tumor tissue and the tissue adjacent to the esophageal squamous cell carcinoma to compare the expression level of HN1L gene in the esophageal squamous cell carcinoma tumor tissue and the tissue adjacent to the esophageal squamous cell carcinoma tumor tissue. Wherein the esophageal squamous carcinoma tumor tissue and the matched paracarcinoma normal esophageal epithelial tissue sample are collected in tumor hospitals of Linzhou city of Henan province
The specific method of immunohistochemical staining is as follows:
(1) tissue section dewaxing and hydrating: placing the tissue slices in a 65 ℃ oven for baking for 2 hours; soaking in xylene for three times, each time for 10 min; gradient alcohol (100%, 95%, 75%50%) for four times, each time for 5 min; then 3% H 2 O 2 Inactivating endogenous peroxidase, soaking for 10min, and soaking in deionized water for 3 min.
(2) The sections were removed together with the beaker and allowed to cool naturally (without taking the sections directly), washed 3 times with PBS for 5min each.
(3) And (3) throwing the slide, wiping liquid around the sliced tissues (taking care to avoid drying the tissues) by using dust-free paper, putting the slide in a wet box, circling the tissues on the slide by using an oily marking pen, dripping 5% BSA (bovine serum albumin) confining liquid on the circled tissues, then putting the slide in the wet box, and incubating the slide for 30min at 37 ℃.
(4) The blocking solution was discarded, HN 1L-specific primary antibody working solution (purchased from Sigma, USA, cat # HPA041908, working concentration: 1:4000 dilution) was added dropwise, and the mixture was placed in a wet box and incubated overnight at 4 ℃. The next day the wet box was removed and left at room temperature for 10min, followed by 3 washes with PBS for 5min each.
(5) Spin-drying, adding secondary antibody working solution (purchased from Dako of Denmark, Cat.: # K5007, Ready-to-use) dropwise onto the circled tissue, placing in a wet box, and incubating at 37 deg.C for 30 min.
(6) PBS wash 3 times, each time for 5 min. The liquid around the sliced tissue is thrown away and wiped dry, the tissue is not dried, the slide is placed in a wet box, DAB working solution prepared in advance is dripped, and color development is controlled under a light mirror. After the color development was completed, the color development was terminated by rinsing with distilled water gently.
(7) And (3) performing hematoxylin counterstaining for 30 seconds, flushing with running water, soaking for 1-3 seconds with the differentiation solution, flushing again with the running water, and bluing the tissue. Finally, dehydration treatment was carried out with 50%, 75%, 95%, 100% gradient alcohol, each for 3 min.
(8) The xylene clarification treatment was repeated 2 times for 5min each time. The gel was sealed with neutral gum and photographed under a light mirror.
Expression of HN1L was assessed by immunohistochemical staining results score, staining intensity score: negative, 0; weak positive; 1; moderate positivity, 2; strong positive, 3. Proportion of HN1L positive cells: < 25%; 1; 25% -50%, 2; 50% -75%, 3; > 75%, 4. Total staining score is staining intensity x positive proportion. And the difference of staining scores of HN1L in esophageal squamous carcinoma tissues and normal esophageal epithelial tissues was counted by using a paired t-test method (bilateral). P <0.05 was considered statistically significant.
Second, experimental results
As shown in FIG. 1B, HN1L protein was expressed at a lower level in the cancer paracsophageal epithelial cells, mainly in the nucleus and cytoplasm of esophageal squamous carcinoma cells.
As shown in fig. 1C and 1D, immunohistochemical staining using an esophageal squamous carcinoma tissue chip (n ═ 229) revealed that the expression level of HN1L protein in tumor tissue was significantly higher than that in paracancerous normal esophageal epithelial tissue (P < 0.001).
Example 3 high expression of HN1L in esophageal squamous carcinoma tissues is associated with poor prognosis in patients
First, experiment method
According to the staining score of HN1L in esophageal squamous carcinoma tissues in example 2, samples were divided into HN1L high expression group and HN1L low expression group by ROC curve method. The correlation between the expression level of HN1L and local invasion of tumor tissues, lymph node metastasis and later stage of tumor is analyzed by a chi-square test method. The Kaplan-Meier method was used to analyze the correlation of HN1L expression level with the total survival time of the patients. P <0.05 was considered statistically significant.
Second, experimental results
The results are shown in fig. 2, and the analysis of correlation of clinical pathological data shows that the esophageal squamous carcinoma tissue up-regulated HN1L is significantly correlated with local invasion of tumor tissue (P <0.001), lymph node metastasis (P <0.05), late stage of tumor (P <0.001) (shown in fig. 2A) and poor prognosis of patients (P ═ 0.038) (shown in fig. 2B), and the difference is statistically significant. The result shows that the high expression of HN1L protein in esophageal squamous carcinoma tissues indicates poor prognosis of patients.
Example 4 establishment of HN1L Stable knockdown esophageal squamous carcinoma cell line by lentivirus-mediated Gene interference technique
First, experiment method
The shRNA-containing lentiviral interference vector psi-LVRU6P (vector purchased from GeneCopoeia, USA, interference sequence: 5'-GGCGTAAGCAGAAACACTAAC-3') was transduced into 293FT tool cells together with three lentiviral packaging vectors pLP1, pLP2 and pLP-VSVG (purchased from Invitrogen, USA) using Lipo3000 transfection reagent (purchased from Dojindo, Japan, Cat.: # H357), virus supernatants were collected 48 hours later and transfected into esophageal squamous carcinoma cells, which were further screened for two weeks using the drug Puromycin (2. mu.g/ml), and HN1L stably knocked-down cell line was established to give K150-shHN1L cells.
Interference negative control group: the same procedure was carried out with the Scramble shRNA vector (purchased from GeneCopoeia, USA) to obtain K150-Scramble cells.
Western blot was used to verify the protein expression levels of HN1L in K150-shHN1L cells and K150-Scramble cells.
Second, experimental results
The results are shown in FIG. 3A, and the stably knocked-down esophageal squamous carcinoma cells (K150-shHN1L) of the prepared HN1L were detected by Western blot, and it was shown that HN1L was knocked down at the protein level compared to the Scramble interference negative control group (K150-Scramble).
Example 5 knockdown of HN1L inhibits metastasis of esophageal squamous carcinoma cells
First, experiment method
(1) Transwell migration experiment
The K150-Scramble cells and K150-shHN1L cells prepared in example 4 in the logarithmic growth phase were each added to a medium containing 0.1% FBS, and starvation-cultured for 24 hours. Trypsinizing the cells, centrifuging at 1000rpm for 5 minutes, resuspending the medium, counting, diluting the cells to 1X 10 in serum-free medium 5 0.5ml, added to the upper chamber and 0.75ml of medium containing 10% FBS added to the lower well. Placing at 37 ℃ and 5% CO 2 Under the conditions, the culture was carried out for 24 hours. After removing non-invaded cells with a small cotton swab, the cells were fixed in alcohol for 10 minutes and stained with crystal violet for 30 minutes. Removing crystal violet by suction, washing with tap water, air drying, and mounting on glass slide. Observation under a microscope and photographing. The 10 fields were picked up randomly and the average number of migrated cells per field was calculated.
(2) Pulmonary metastasis experiment of tail vein of nude mouse
Female BALB/c nude mice, 4-5 weeks old,the weight of the animal is 18-20 g, and the animal is purchased from the center of medical laboratory animals in Guangdong province. Mice were raised in an SPF-level environment, acclimated for one week. The cells K150-Scramble and K150-shHN1L prepared in example 4 were collected at logarithmic phase, trypsinized, centrifuged at 1000rpm for 5 minutes, resuspended in medium, and counted. The injection amount is 5X 10 5 Cells/100. mu.l. First, nude mice were anesthetized with isoflurane gas and the limbs were fixed with tape. The cell suspension was injected slowly from the tail vein of the mouse using a 1ml insulin needle. After 2 months, the mice were sacrificed by cervical dislocation, lung tissues were surgically excised and fixed with 10% neutral formalin solution, paraffin-embedded, tissue sectioned and hematoxylin-eosin stained, and finally observed under a microscope to confirm the presence of tumor cells.
Second, experimental results
The results of the in vitro Transwell migration experiment (as shown in FIG. 3B) showed that there were fewer tumor cells migrating through the Transwell chamber after knockdown of HN1L (K150-shHN1L) compared to the control cells (K150-Scramble).
The results of the in vivo nude mouse tail vein lung metastasis experiments (as shown in FIG. 3C) show that esophageal squamous carcinoma cells (K150-shHN1L) after knockdown of HN1L were found to form fewer nodules in the lung compared to the control cells (K150-Scramble).
The in vivo and in vitro experiment results show that the knock-down HN1L obviously inhibits the transfer capacity of esophageal squamous cell carcinoma cells.
Example 6 knockdown of HN1L inhibits growth of esophageal squamous carcinoma cells
First, experiment method
(1) In vitro CCK-8 cell proliferation assay
The cells K150-Scramble and K150-shHN1L prepared in example 4 were collected at logarithmic phase, trypsinized, centrifuged at 1000rpm for 5 minutes, the DMEM complete medium was resuspended, counted, the cell suspension was diluted appropriately and added to a 96-well plate (1000 cells/well) at 100. mu.l/well. Each group was provided with 4 parallel holes at 37 ℃ and 5% CO 2 The culture was continued for 3 days under the conditions that 10. mu.l of CCK-8 solution (purchased from Dojindo, Japan, cat # CK04) was added at the same time each day, the culture was continued for 2 hours, and after complete color development, enzyme-linked immunosorbent assay was performedThe detector measures the OD per well at a wavelength of 450 nm. Cell growth curves were plotted against the OD values of the cells per well.
(2) Subcutaneous tumor formation experiment of nude mice
Female BALB/c nude mice, 4-5 weeks old, weighing 18-20 g, were purchased from Guangdong province medical laboratory animal center. Mice were raised in an SPF-level environment, acclimated for one week. The mice were inoculated with the K150-Scramble and K150-shHN1L cell suspensions prepared in example 4 in the logarithmic growth phase on the left and right sides of the back, respectively. The injection amount is 4X 10 6 Cells/100. mu.l. After four weeks the mice were sacrificed, the tumor tissue was stripped off and the tumors weighed on an electronic balance.
Immunohistochemical staining: the procedure was as in example 2. Ki 67-specific antibodies were purchased from Abcam, usa under the cat number: # ab16667, working concentration: 1:400.
Second, experimental results
In vitro CCK8 cell proliferation assay (as shown in FIG. 4A) showed that the proliferation rate of cells (K150-shHN1L) was lower after knockdown of HN1L than that of control cells (K150-Scramble).
In nude mice subcutaneous transplantable tumor experiments (as shown in FIG. 4B), it was found that the weight of the transplantable tumor formed by esophageal squamous carcinoma cells (K150-shHN1L) after knockdown of HN1L was less than that of the transplantable tumor formed by control cells (K150-Scramble) (P < 0.05).
Immunohistochemical staining analysis (as shown in figure 4C) further confirmed the low proportion of proliferating cells (Ki67 positive) in the transplanted tumors after knockdown of HN 1L.
The in vitro and in vivo experiment results show that the proliferation capacity of esophageal squamous cell carcinoma cells is obviously reduced after HN1L is knocked down.
Example 7 knockdown of HN1L increases sensitivity of esophageal squamous carcinoma cells to docetaxel
First, experiment method
(1) In vitro CCK8 cell proliferation assay
The K150-Scramble and K150-shHN1L cells prepared in example 4 were treated with varying concentration gradients of docetaxel. After 24 hours, the proliferation activity of the cells was examined using CCK-8 reagent. The experimental procedure was as in example 6.
(2) Nude mice subcutaneous transplantation tumor and drug experiment
The K150-Scramble and K150-shHN1L cells prepared in example 4 were subcutaneously implanted in nude mice at an injection rate of 4X 10 6 Cells/100. mu.l. After 2 weeks of tumor cell injection, docetaxel (purchased from Ningdanshengyo group GmbH, China at a dose of 3mg/kg mouse body weight) was administered intraperitoneally by a sterile syringe once every three days for four times. One week after the end of treatment cervical dislocation mice were sacrificed. The tumor tissue is stripped by operation, and the tumor is weighed by an electronic balance.
Second, experimental results
The results are shown in fig. 5, the first-line chemotherapeutic drug docetaxel of esophageal squamous carcinoma is used for treating HN1L to knock down stable strains and control cells, and in vitro CCK8 cell proliferation experiments show that the knock-down HN1L can improve the sensitivity of tumor cells to docetaxel drugs (as shown in fig. 5A). Nude mice subcutaneous transplantations and drug experiments further confirmed that silencing HN1L reversed the resistance of tumor cells to docetaxel drugs (as shown in fig. 5B).
Example 8 knockdown of HN1L increases the therapeutic efficacy of cisplatin on esophageal squamous carcinoma cells
First, experiment method
The K150-Scramble and K150-shHN1L prepared in example 4 were implanted subcutaneously in nude mice at an injection rate of 4X 10 6 Cells/100. mu.l. Cisplatin (purchased from Jiangsu Haisen pharmaceutical industry group, Inc. in China, in an amount of 5mg/kg of mouse body weight) was administered intraperitoneally using a sterile syringe 2 weeks after injection of tumor cells, once every three days for four administrations. One week after treatment the mice were sacrificed by cervical dislocation. The tumor tissue is stripped by operation, and the tumor is weighed by an electronic balance.
Second, experimental results
The results are shown in figure 6, the results of nude mouse subcutaneous transplantable tumor and drug experiment show that the transplanted tumor from K150-shHN1L treated by cisplatin drug (DDP) is lighter in weight, and indicate that silencing HN1L can enhance the killing effect of cisplatin drug on esophageal squamous cell carcinoma cells.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and that those skilled in the art can make other variations or modifications on the basis of the above description and idea, and that all embodiments are neither necessary nor exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. The application of the reagent for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in preparing an esophageal squamous carcinoma diagnostic kit, wherein the HN1L gene, HN1L mRNA or/and HN1L protein expression level of patients with esophageal squamous carcinoma is higher than that of patients without esophageal squamous carcinoma.
2. The esophageal squamous carcinoma diagnostic kit according to claim 1, wherein the esophageal squamous carcinoma diagnostic kit is a kit for diagnosing tumor tissue invasion, and HN1L gene, HN1L mRNA or/and HN1L protein are highly expressed in a patient, while tumor tissue invasion is severe in a relatively lowly expressed patient.
3. The esophageal squamous carcinoma diagnostic kit according to claim 1, wherein the esophageal squamous carcinoma diagnostic kit is a kit for diagnosing lymph node metastasis, and HN1L gene, HN1L mRNA and/or HN1L are highly expressed in patients, and relatively lowly expressed in patients, lymph node metastasis is severe.
4. The esophageal squamous carcinoma diagnostic kit according to claim 1, wherein the esophageal squamous carcinoma diagnostic kit is a kit for diagnosing staging conditions, and patients with high expression of HN1L gene, HN1L mRNA or/and HN1L protein are relatively low-expressing patients and are staged later.
5. The application of a reagent for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in preparing an esophageal squamous carcinoma prognosis prediction kit is disclosed, wherein patients with HN1L gene expression, HN1L mRNA expression or/and HN1L protein high expression are relatively low expression patients, and the prognosis is poor.
6. The application of a reagent for detecting HN1L gene expression, HN1L mRNA expression or/and HN1L protein expression in preparing a sensitivity detection kit for chemotherapy of patients with esophageal squamous carcinoma is characterized in that patients with HN1L gene expression, HN1L mRNA expression or/and HN1L protein high expression are relatively low-expression patients, and the sensitivity of chemotherapy is poor.
7. The use of claim 6, wherein the chemotherapeutic agent is docetaxel or cisplatin.
The application of the inhibitor of HN1L gene expression, the inhibitor of HN1L mRNA expression or/and the inhibitor of HN1L protein expression in the preparation of the synergist of the esophageal squamous carcinoma medicines or the synergist of the esophageal squamous carcinoma chemotherapeutic medicines.
9. The use of claim 8, wherein the chemotherapeutic agent is docetaxel or cisplatin.
10. An esophageal squamous carcinoma medicament is characterized by comprising an inhibitor of HN1L gene expression, an inhibitor of HN1L mRNA expression and/or an inhibitor of HN1L protein expression, and preferably further comprising a chemotherapeutic medicament, wherein the chemotherapeutic medicament is docetaxel or cisplatin.
CN202210550398.0A 2022-05-20 2022-05-20 Application of reagent for detecting HN1L expression in preparation of esophageal squamous carcinoma diagnostic kit Pending CN115044673A (en)

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