WO2012175052A1 - 逆转或降低食管癌放疗抗性的增敏剂、筛选方法及其用途 - Google Patents

逆转或降低食管癌放疗抗性的增敏剂、筛选方法及其用途 Download PDF

Info

Publication number
WO2012175052A1
WO2012175052A1 PCT/CN2012/077434 CN2012077434W WO2012175052A1 WO 2012175052 A1 WO2012175052 A1 WO 2012175052A1 CN 2012077434 W CN2012077434 W CN 2012077434W WO 2012175052 A1 WO2012175052 A1 WO 2012175052A1
Authority
WO
WIPO (PCT)
Prior art keywords
akr1c3
esophageal cancer
gene
kyse
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2012/077434
Other languages
English (en)
French (fr)
Inventor
周德敏
高献书
熊伟
赵静
张礼和
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Peking University
Peking University First Hospital
Original Assignee
Peking University
Peking University First Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peking University, Peking University First Hospital filed Critical Peking University
Publication of WO2012175052A1 publication Critical patent/WO2012175052A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators

Definitions

  • the present invention relates to the field of biotechnology and immunotherapy, and in particular to sensitizers, screening methods and uses thereof for reversing or reducing the resistance of esophageal cancer radiotherapy. Further, it relates to a gene silencing agent, a silencing method and a use thereof for a radiotherapy resistance gene of esophageal cancer.
  • the present invention can effectively achieve the sensitization effect of radiotherapy by silencing the selected specific resistance gene, thereby enabling better individualized treatment and providing a more effective treatment plan for the treatment of esophageal cancer. Background technique
  • Esophageal cancer is a common malignant tumor and is the sixth cause of cancer-related death worldwide [Enzinger PC & Mayer RJ. Esophageal cancer. N Engl J Med 2003; 349: 2241 - 2252] ⁇ known radiotherapy is esophageal cancer One of the main treatments, but the local failure rate is as high as 50% - 55%, even if the dose of radiotherapy or chemotherapy is increased, the rate of local control cannot be improved [Cooper J et al, Radiation Therapy Oncology Group for the Chemoradiotherapy of locally advanced esophageal cancer: Long -term fol low-up of a prospective randomized trial (RT0G 85-01) .
  • the inventors conducted various research work, and a lot of work has been done on the search for resistance-sensitive genes.
  • AKR1C3 was expressed in esophageal cancer cells, normal esophageal mucosal cells, interstitial cells and small blood vessel epithelium.
  • the expression of normal esophageal mucosal cells was significantly lower than that of highly differentiated esophageal squamous carcinoma cells;
  • subsequent studies have shown that AKR1C3 may provide a new target for radiosensitizing drugs for esophageal cancer, especially for highly differentiated squamous cell carcinoma of the esophagus.
  • AKR1C3 Homo sapiens aldo-keto reductase family 1, member C3 (3 - alpha hydroxysteroid dehydrogenase, type II)
  • Genbank number of the sequence was found to be NM-003739, and its complete sequence is as follows:
  • AKR1C3 protein is closely related to radioresistance
  • the AKR1C family is a 37 kDa soluble protein that can be reduced to a hydroxyl group by NAD (P) (H)-mediated hydrogen ion transport.
  • AKR1C3 is a member of the aldehyde ketone reductase superfamily.
  • AKR1C has four subtypes, namely AKR1C1, AKR1C2, AKR1C3 and AKR1C4.
  • AKR1C3 has more than 86% of the sequences homologous to the remaining three AKRs.
  • AKR1C3 has 3 oc -HSD, ⁇ ⁇ -HSD, 20 -HSD, and prostacyclin (PG) F synthetase activity, which catalyzes the metabolism of androgens, estrogens, prostaglandins, prostacyclin and exogenous substances.
  • PG prostacyclin
  • AKR1C3 mRNA was first discovered and expressed in the mammary gland and prostate, and subsequent expression of the gene was found in the liver, lung, small intestine, adrenal gland, brain, uterus and testis.
  • AKR1C3 has been shown to be dysregulated in many types of cancer, including osteophyte hyperplasia syndrome (MDS, refractory anemia), endometrial cancer, and up-regulation in lung cancer.
  • MDS osteophyte hyperplasia syndrome
  • endometrial cancer endometrial cancer
  • up-regulation in lung cancer up-regulation in lung cancer.
  • AKR1C3 in esophageal cancer has not been reported yet.
  • the inventors constructed a series of vectors targeting AKR1C3 as candidate drugs to test the feasibility of this screening method at the cellular and animal levels. Specifically, the expression of AKR1C3 was silenced by the constructed vector, and the silenced cells were subjected to radioactivity test to observe the relationship between the gene silencing and radioresistance.
  • the candidate drug used in the examples is an antisense molecule, and in fact The candidate drug is detected by various known compound libraries or the like.
  • Other compounds capable of expressing the expression of AKR1C3, such as protein nucleic acids, etc., can also be screened.
  • the inventors established a cell screening model and an animal screening model, and selected three genes known to have a targeting effect on the AKR1C3 gene as candidate drugs, and silently screened AKR1C3 in animals, and successfully silenced nude mice.
  • the radiotherapy resistance was tested to verify the reliability of the screening model and to test the sensitization effect of the selected target sequence.
  • the present invention provides a target for sensitization therapy for esophageal cancer radiotherapy resistance - the AKR1C3 gene, and a gene drug for sensitization therapy for esophageal cancer radiotherapy resistance, which is the AKR1C3 gene
  • a gene drug for sensitization therapy for esophageal cancer radiotherapy resistance which is the AKR1C3 gene
  • An expression inhibitor, or a silencing agent, preferably, the sensitizer is an antisense nucleic acid.
  • it contains a pharmaceutically acceptable adjuvant.
  • the invention provides a method of sensitizing therapy for radiotherapy resistance of esophageal cancer comprising the step of silencing or inhibiting expression of the AKR1C3 gene.
  • the individual to be treated is subjected to an esophageal cancer cell classification test, preferably said step is performed on an individual having an esophageal squamous cell carcinoma, and further preferably said step is performed on a highly differentiated individual of esophageal squamous cell carcinoma.
  • the step is to administer an effective amount of an AKR1C3 gene silencing agent or an AKR1C3 gene inhibitor to an individual in need thereof.
  • the present invention provides a non-therapeutic purpose for the treatment of esophageal cancer radiotherapy Screening of cells, which includes the step of subjecting the cells to AKR1C3 gene silencing or expression inhibition.
  • the cell is ex vivo, or the cell is from an individual of esophageal squamous cell carcinoma, preferably from a highly differentiated individual of esophageal squamous cell carcinoma.
  • the invention provides a non-therapeutic purpose for screening a radiotherapy resistant animal for esophageal cancer comprising the step of subjecting the animal to AKR1C3 gene silencing or expression inhibition.
  • the animal may be a rat, a mouse or a rabbit or the like which is loaded with esophageal cancer radiotherapy-resistant cells.
  • the step of using AKR1C3 gene silencing or expression inhibition in the present invention includes administration of a drug, such as a gene therapy drug. It can also be silenced by gene knockout techniques and the like.
  • the invention provides the use of an AKR1C3 gene expression inhibitor or a silencing agent for the preparation of a gene therapy for sensitization therapy for esophageal cancer radiotherapy resistance, wherein the inhibitor may be methyl jasmonate (Methyl) Jesmonate, MeJ); Jasmonate, JA.
  • the inhibitor may be methyl jasmonate (Methyl) Jesmonate, MeJ); Jasmonate, JA.
  • the silencing agent refers to a compound capable of not expressing the AKR1C3 gene in vitro or in vivo, and may be a nucleic acid fragment, a protein, a saccharide or the like, or may be a chemical small molecule.
  • the inhibitor refers to a compound which is capable of not expressing or underexpressing the AKR1C3 gene in vitro or in vivo, and may be a nucleic acid fragment, a protein, a saccharide or the like, or may be a chemical small molecule.
  • the target of the action may be the AKR1C3 gene itself, or a regulatory element of AKR1C3 gene expression, such as a promoter.
  • the present invention provides an initial screening method for an esophageal cancer radiosensitivity sensitizer, comprising:
  • a drug candidate capable of down-regulating or disappearing the expression of the AKR1C3 gene was identified as a drug having the potential of a radiosensitivity sensitizer for esophageal cancer.
  • subsequent animal testing or the like can be performed for verification.
  • a host cell which highly expresses the AKR1C3 gene means that the expression amount of the host cell which is not transfected with the AKR1C3 gene is high.
  • the present invention provides a cell level screening method for an esophageal cancer radiosensitivity sensitizer, which comprises
  • a drug candidate capable of down-regulating or disappearing the expression of the AKR1C3 gene while maintaining the survival of esophageal cancer cells can be judged to be a radiosensitivity sensitizer for esophageal cancer.
  • esophageal cancer cells with high expression of the AKR1C3 gene are highly expressed in radiation-sensitive esophageal cancer cells, such as the KYSE-170R cell line.
  • the appropriate lower contact of i1 ⁇ 2 means that the normal growth of esophageal cancer cells with high expression of AKR1C3 gene is in addition to the candidate drug, and other growth conditions are consistent with conventional growth conditions.
  • the invention also provides an animal level screening method for an esophageal cancer radiosensitivity sensitizer, which comprises
  • a drug candidate capable of down-regulating or disappearing the expression of the AKR1C3 gene and making the tumor smaller or disappearing can be judged as a radiosensitivity sensitizing drug for esophageal cancer.
  • esophageal cancer cells that highly express the AKR1C3 gene are highly expressed in eradiometric esophageal cancer cells, such as the KYSE-170R cell line.
  • the animal may be a nude mouse or other test animal.
  • the administration method may be oral administration, intravenous infusion, or intratumoral injection.
  • oral administration intravenous infusion, or intratumoral injection.
  • Figure 1 shows the results of comparison of differentially expressed genes between the radioresistant cell line KYSE-170R and the non-radioactive cell line KYSE-170. among them,
  • Figure I-A Comparison of KYSE-170R and KYSE-170 cell line chip differences. It was found that there were 945, 733 and 1232 differentially expressed genes at 8 hours after irradiation and 24 hours after irradiation. Among them, AKR1C3 has an I l lumine difference value of > 50 at three time points. Among them, I l lumine difference value > 20 is considered to be 1 ⁇ 2 due to up-regulation; I l lumine difference value ⁇ -20 is considered to be up-regulated.
  • FIG. 1B Application of qRT-PCR on the mRNA 7j level, as shown by the graph, the expression of AKR1C3 was significantly different at the mRNA level. Compared with the parental KYSE-170, the expression of AKR1C3 in the resistant KYSE-170R cells was up-regulated by 9.39, 5.94 and 9.20 times, respectively, at Oh, 8h and 24h after irradiation.
  • Figure 1-C Western blot results, which confirmed that the expression of AKR1C3 protein in the non-irradiated radiation-resistant cell line KYSE-170R was significantly higher than that of the parental cell line KYSE-170 (*P ⁇ 0.05).
  • FIG. 1 Radiosensitivity test results after overexpression of AKR1C3 in AKR1C3 silenced KYSE-170R cells and parental KYSE-170 cells. among them
  • Figure 2-A shows the results of qRT-PCR, which shows that the AKR1C3 silencing efficiency is 60%.
  • Figure 2 - B, C shows the results of western blot, which confirmed that AKR1C3 expression was significantly down-regulated at the protein level.
  • FIG. 2 - D E: Radiation resistance assay of AKR1C3 silenced KYSE-170R cells.
  • the results of colony formation assay showed that the clone formation rate of scramble-shRNA-KYSE-170R and AKR1C3-shRNA-KYSE-170R cells was not observed when not irradiated. Significant differences indicate that AKR1C3 silencing does not affect KYSE-170R proliferation. After 2, 4, 8 Gy irradiation, the number of clones of KYSE-170R cells silenced by AKR1C3 was significantly reduced, and the radiosensitivity was significantly enhanced.
  • FIG. 2- F, G Western blot verification after overexpression of AKR1C3 in parental KYSE-170 cells that are relatively sensitive to radiation.
  • the AKR1C3 overexpression plasmid was transiently transfected into the parental cell line KYSE-170 and transiently transfected with the scramble plasmid as a control. Results Western blot confirmed that the overexpression effect was significant.
  • FIG. 2-H I: Clonal formation assay to detect radioresistance of KYSE-170 cells overexpressing AKR1C3. Results There was no significant difference in the rate of clone formation between scramble-KYSE-170 and AKR1C3+ KYSE- 170 cells, indicating that AKR1C3 overexpression did not affect KYSE-170 proliferation. After 2, 4, 8 Gy irradiation, the number of clones of KYSE-170 cells overexpressed by AKR1C3 was significantly increased, and the radioresistance was significantly enhanced. I China represents the AKR1C3+ KYSE-170 cell group; * represents the scramble-KYSE-170 cell group.
  • Figure 3 Detection of target gene sink in a nude mouse tumor model; 11 ⁇ 2 effect on tumor radiation resistance.
  • Figure 3-A When the diameter of the tumor reaches 6-8, the second and fourth groups give a single partial exposure of 15Gy of the irradiation dose.
  • Figure 3- B Based on the observations, plot the tumor volume of nude mice. The curve suggests that after receiving radiotherapy, the tumors formed by AKR1C3-shRNA-KYSE-170R cells stably silencing AKR1C3 were more significantly reduced than those inoculated with scramble-shRNA-KYSE-170R cells; When the tumor growth rate is not significantly different different.
  • * represents s cr amb 1 e-shRNA-KYSE-17 OR cell group; country represents AKR1C3- shRNA-KYSE- 170R cell group; ⁇ represents s cr amb 1 es hRNA-KYSE-17 OR cell + radiotherapy group; + AKR1C3 - shRNA- KYSE- 170R cells + radiation therapy group.
  • Figure 3- C Results of immunohistochemistry of AKR1C3 protein in nude mice that were not receiving radiotherapy.
  • the expression of AKR1C3 protein in the scramble-shRNA-KYSE-170R group was significantly higher than that in the AKR1C3-shRNA-KYSE-170R group.
  • Figure 3-D The results of immunohistochemical detection of AKR1C3 protein in tumor tissues of nude mice that did not receive radiotherapy were AKR1C3-shRNA-KYSE-170R group.
  • Figure 3- F HE staining of tumor tissues of nude mice after radiotherapy showed that AKR1C3-shRNA-KYSE-170R cells formed a large amount of scar tissue in the tumor tissue, leaving only a small amount of tumor cells.
  • Figure 3- E HE staining results of tumor tissues of nude mice remaining after radiotherapy showed that there were a large number of tumor cell residues in the tumor tissue formed by scramble-KYSE-170R cells as a control.
  • Figure 3-G The results of qRT-RCR were obtained for each of the scramble-KYSE-170R group and the third group AKR1C3-shRNA-KYSE-170R group.
  • Figure 4 Retrospective analysis of the expression of AKR1C3 in the pathological tissues of patients with esophageal cancer by immunohistochemistry. among them
  • Figure 4-A Expression in pathological specimens of 6 patients with poorly differentiated squamous cell carcinoma of the esophagus
  • Figure 4-B Expression in pathological specimens of 6 patients with highly differentiated squamous cell carcinoma of the esophagus
  • Figure 4-C Normal esophagus adjacent to the cancer Expression in mucosal cell pathological specimens.
  • Figure 4- D Comparison of the results of Figure 4A-C.
  • CGMCC No: 4936 classified as human esophageal squamous cell carcinoma cell line KYSE-17 OR). Both of the above cells were donated by the Department of Thoracic Oncology and Radiation Therapy of MD Anderson Cancer Center, USA.
  • the above cell lines were cultured in high glucose DMEM medium (purchased from Invitrogen) using high glucose 1640 medium (purchased from Invitrogen) and HEK293T cells (provided by the State Key Laboratory of Peking University School of Pharmacy). 10% inactivated fetal bovine serum (FBS, purchased from HyClone) and penicillin (50- ⁇ /mL each) were added to the supernatant. Culture conditions: 37 incubator, 5% C0 2 .
  • the number of cells was counted using a Backman Z1 cell counter (purchased from Backman, USA). At the Department of Radiation Oncology, Peking University First Clinical Hospital, the cells were irradiated with 6MV-XH
  • PCR primers were designed by Primer 3.0 software and specific for Blast.
  • Target sequence AKR1C3 (GenBank: M-003739, SEQ ID NO: 1)
  • Downstream primer 5 tgagttttccaaggctggtc 3, (SEQ ID NO: 3);
  • Target sequence ⁇ -acting
  • Upstream primer 5, agcgagcatcccccaaagtt 3, (SEQ ID NO: 4); downstream primer: 5, gggcacgaaggctcatcatt 3, (SEQ ID NO: 5).
  • PCR reactions were performed using a Roche 480 PCR machine (Roche, USA), specific conditions: first cycle (95 - 10 minutes); second cycle (95 - 30 seconds, 60 - 30 seconds, 72 - 30 seconds) x40.
  • KYSE-170 and KYSE-170R cells in logarithmic growth phase were washed twice with cold PBS. Depending on the amount of cells, appropriate amount of protein lysate (RPIA purchased from Applygene) was added and lysed on ice bath for 30 min. 4* ⁇ Centrifugation at 14000 rpm for 30 min, discard the lower layer of sediment, and collect the supernatant to obtain whole cell protein. Proteins were quantified using the Bio-Rad Protein Quantification Kit (Bio- Rad, Hertfordshire, United Kingdom).
  • the protein was separated by electrophoresis on a 12% SDS-polyacrylamide gel, and the protein on the gel was transferred to an ECL Chemi luminescent substrate (Amersham Pharmacia Biotech, Piscataway, NJ, USA, containing 5% skim milk powder and 0.1% spit).
  • the warm blocking solution was blocked for 1 hour at room temperature.
  • the primary antibody was incubated overnight, and the sputum was washed 4 times with TBST for 5 min each, then incubated for 2 hours at room temperature in the secondary antibody.
  • TBST was washed 4 times for 15 min each.
  • Human AKR1C3 monoclonal antibody (1: 1000) was purchased from Sigma; rabbit anti-human GAPDH monoclonal antibody (1: 1000) was purchased from Santa Cruza.
  • shRNA lentiviral vector vector silencing AKR1C3 Construction of shRNA lentiviral vector vector silencing AKR1C3 and screening of the best shRNA vector
  • the inventors selected a radiotherapy-resistant KYSE-170R cell line as a screening cell, and constructed a series of vectors targeting AKR1C3 as a drug candidate.
  • the AKR1C3 expression was silenced by the constructed vector, and the silenced cells were subjected to radiological examination to observe the relationship between the gene silencing and radioresistance.
  • the Lent i virus vector is a gene therapy vector based on HIV-1 (human immunodeficiency type I virus). It distinguishes between a general retroviral vector, which is infectious to both dividing cells and non-dividing cells.
  • the vector can efficiently integrate a foreign gene into the host chromosome to achieve persistent expression.
  • the inventor applied the optimized remodeling lentiviral vector PSD31 (ZJ J et al., A more eff icient RNAi inducible system for maintain regulation of gene expression in mammalian cells and xenograft animals. RNA. 2007 Aug; 13 (8) : 1375-83 . . , carrying a shRNA sequence that can target silencing AKR1C3, transfecting cells, and silencing the gene of interest.
  • shRNA primers were synthesized by Beijing Huada Gene.
  • the human U6 promoter was used to initiate the transcription process.
  • the human U6 promoter sequence is as follows:
  • downstream primer sequence of scramble is as follows:
  • shRNA (0) This corresponds to the base position of SEQ ID NO: 1: 148- 168
  • shRNA (1) which corresponds to the position of SEQ ID NO: 1 693-983:
  • shRNA (2) which corresponds to the base position of SEQ ID NO: 1 372-390:
  • GTTTCGTCCTTTCCAC-3' (SEQ ID NO: 10) .
  • shRNA (0) - (2) the sinking of these three silencing vectors; the upstream primers of the I ⁇ column are:
  • the PCR product was ligated to the pGEM T easy vector vector using the T4 DNA Ligation Kit (NEB, Beverly, MA, USA) and sent to China.
  • the company confirmed the correctness of the DNA sequence by DNA sequencing.
  • the correct TA cloning plasmid was sequenced and transiently transfected into KYSE-170R cells according to the mega tans 1. 0 (0rigen) transfection kit step. Then perform Western blotting to select.
  • HEK 293T cells purchased from Invitrogen
  • PSD31-AKR1C3-shRNA and viral packaging helper plasmids VSVG, PRSV and PMDL plasmids purchased from Invi trogen
  • PRSV viral packaging helper plasmids
  • PMDL plasmids purchased from Invi trogen
  • co-transfection of 4 plasmids is the virus packaging process.
  • the PSD31- AKR1C3-shRNA plasmid is the core part of the virus formation
  • the VSVG, PRSV and PMDL virus packaging helper plasmids are auxiliary core plasmids, which function as a helper virus envelope formation, cell vomiting virus and the like.
  • the inventors obtained an effective AKR1C3 expression silencing agent by screening with an effective cell model.
  • the colony formation assay is a classic experiment to evaluate the effects of radiation therapy.
  • the inventors will detect the number of clones of the AKR1C3 gene stably silenced cell line at different irradiation doses and the unsilenced cell line scramble-shRNA-KYSE-170R (this line was established simultaneously with AKRlC3-shRNA-KYSE-170R as a negative control).
  • scramble-KYSE-170R is linked to the A t function gene scramble. See the difference in establishing a stable silencing AKR1C3 cell line.
  • the number of clone formation reflects the sensitivity of this cell line to radiation therapy. The more clones are formed, the lower their sensitivity and the stronger the resistance.
  • the scramble-KYSE-170R and AKRlC3-shRNA-KYSE-170R cells were administered to the logarithmic growth phase in the order of (2, 4, 8 Gy), followed by trypsin digestion, and the cell counter Vi-CELL (purchased from BECKMAN) ) Count the cells. Adjust the number of cells inoculated according to different irradiation measurements.
  • the same amount of scramble-KYSE-170R and AKRlC3-shRNA-KYSE-170R cells were seeded on six, cultured for 7-10 days, removed, fixed, crystal violet staining (Sigma Chemical Co.), application The number of clones counted (containing 50 or more cells as one clone). The number of clones when the corresponding cells were not irradiated was counted to correct the inoculation rate and to enhance comparability.
  • Example 1 the inventors selected the esophageal squamous cell carcinoma resistant cell line (KYSE-170R) and the parental cell line (KYSE-170) with the same genetic background. Differences in gene expression at different time points after irradiation and irradiation, and dynamic analysis of gene expression changes before and after irradiation, to a certain extent, avoid the leakage selection caused by the initiation of radiation resistance gene expression by irradiation. Based on the fold difference, GO analysis and Pathway analysis, it is speculated that 10 genes such as AKR1C3, which are up-regulated in the radiation-resistant cells, may be involved in the formation of radiation resistance. Further in vitro validation results indicate that AKR1C3 gene and esophageal cancer radiation Resistance related.
  • the combined results of each experiment can confirm that the AKR1C3 gene is a differentially expressed gene between the radioresistant cell KYSE-170R and the radiosensitized cell KYSE-170, and silencing the AKR1C3 gene in the radioresistant cell KYSE-170R leads to a decrease in radioactivity of the cell.
  • the radiosensitive cell KYSE-170 i ⁇ up to the AKR1C3 gene can increase the radioresistance of this cell, demonstrating that the AKR1C3 gene is an important gene involved in radioresistance.
  • Example 3 Establishment of animal screening models and sensitization of gene silencing methods
  • mice aged 4-6 weeks were randomly divided into 4 groups (5 in each group): group 1: scramble-KYSE-170R group; group 2: scramble-KYSE-170R+ radiotherapy group; group 3: AKR1C3- shRNA- KYSE- 170R group; Group 4: AKR1C3- shRNA- KYSE- 170R+ radiotherapy group.
  • group 1 scramble-KYSE-170R group
  • group 2 scramble-KYSE-170R+ radiotherapy group
  • group 3 AKR1C3- shRNA- KYSE- 170R group
  • Group 4 AKR1C3- shRNA- KYSE- 170R+ radiotherapy group.
  • the human esophageal cancer cells scramble-KYSE-170R and AKRlC3-shRNA-KYSE-170R in the logarithmic growth phase were trypsinized, washed with PBS, and resuspended to a concentration of 2 x 107
  • the tumor volume curve of nude mice was drawn (see Figure 3-B).
  • the curve suggests that after receiving radiotherapy, compared with the inoculation scramble-KYSE-170R, the tumors formed by AKR1C3-shRNA-KYSE-170R cells stably silencing AKR1C3 are more significant, while the two groups are not receiving radiotherapy. There was no significant difference in tumor growth rate.
  • HE staining of tumor tissues of nude mice after radiotherapy revealed that only a small amount of tumor cells remained in the tumor tissue formed by AKRlC3-shRNA-KYSE-170R cells (see Figure 3-F), while the scramble of the control - There are a large number of tumor cell residues in the tumor tissue formed by KYSE-170R cells (see Figure 3-E).
  • AKR1C3-shRNA-KYSE-170R group Three groups of scramble-KYSE-170R group and group 3 AKR1C3-shRNA-KYSE-170R group were subjected to qRT-RCR (see Figure 3-G) and western blot (see Figure 3-H, I). The expression of AKR1C3 was further different between the two groups. The results suggest that the expression of AKR1C3 in the scramble-KYSE-170R group is indeed higher than that in the AKR1C3-shRNA-KYSE-170R group (* ⁇ 0.05).
  • Example 4 Statistical analysis of the expression of AKR1C3 in pathological tissues of patients with esophageal cancer
  • AKR1C3 in the pathological tissues of patients with esophageal cancer by immunohistochemistry, the inventors found that it is expressed in esophageal cancer cells (see Figure 4 - ⁇ , ⁇ ), normal esophageal mucosal cells (see Figure 4- C) Medium.
  • the study group selected 6 cases of esophageal well-differentiated squamous cell carcinoma patients (see Figure 4 - ⁇ ); 6 cases of esophageal poorly differentiated squamous cell carcinoma patients (see Figure 4 - ⁇ ) and adjacent normal esophageal mucosal cell pathology specimens (see Figure 4- C) Perform a retrospective analysis.
  • AKR1C3 expression was significantly increased in highly differentiated esophageal squamous cell carcinoma, further indicating that high expression of AKR1C3 is associated with radiation resistance of esophageal squamous cell carcinoma.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供逆转或降低食管癌放疗增敏剂、筛选方法及其用途，其中所述增敏剂为AKR1C3基因表达抑制剂或沉默剂。通过对AKR1C3基因的沉默，能够有效地实现放疗的增敏效果，进而能够更好地进行个体化治疗，为食管癌的治疗提供更有效的治疗方案。

Description

逆转或降低食管癌放疗抗性的增敏剂、
ffiffl方法及其用途 发明领域
本发明涉及生物技术和免疫治疗领域，具体而言涉及逆转或降 低食管癌放疗抗性的增敏剂、 筛选方法及其用途。 进一步地， 涉及 对食管癌放疗抗性基因的基因沉默剂、 沉默方法及其用途。 本发明 通过对选择的特定抗性基因的沉默， 能够有效地实现放疗的增敏效 果， 进而能够更好地进行个体化治疗， 为食管癌的治疗提供更有效 的治疗方案。 背景技术
食管癌是常见的恶性肿瘤，在全世界是第六位癌症相关的死亡 原因 [Enzinger PC & Mayer RJ. Esophageal cancer. N Engl J Med 2003; 349: 2241 - 2252] β 已知放射治疗是食管癌的主要治疗手段 之一， 但局部失败率高达 50%- 55%， 即使增加放疗或化疗剂量仍然 不能改善局控率 [Cooper J 等人， Radiation Therapy Oncology Group for the Chemoradiotherapy of locally advanced esophageal cancer: Long-term fol low-up of a prospective randomized trial (RT0G 85-01) . JAMA 1999; 281: 1623 - 1627; Minsky B等人， INT 0123 (Radiation Therapy Oncology Group 94-05) phase III trial of combinedmodal ity therapy for esophageal cancer: High-dose versus standard-dose radiation therapy. J Cl in Oncol 2002; 20: 1167 - 1174] β
目前研究发现放疗局部失败除了与肿瘤乏氧、剂量限制性毒性 降低照射量等因素有关外， 与放射抵抗密切相关。 可以说放射抵抗 是放疗后局部复发的主要原因。体内食管癌瘤中某些基因的表达状 态直接影响着癌细胞的放射抗性 [K Fukuda 等人， Differential gene expression prof iles of radioresistant oesophageal cancer cel l l ines establ ished by continuous fractionated irradiation. British Journal of cancer, 2004; 91 (8) : 1543-1550]。
对局部失败率高达 50%- 55%的这部分患者人群， 有必要寻找与 食管癌的放疗抗性相关基因，并以之为靶点获得能够增敏放疗抗性 基因的增敏剂， 借助这种筛选抑制或者消除放疗抗性， 进而为这部 分食管癌患者提供个体化治疗， 提高患者的生存率。 发明内容
为了实现上述发明目的， 发明人进行了多方面的研究工作， 先 在抗性敏感基因搜索方面进行了大量工作。
主要包括：应用包含 48000个探针的 I llumine- 6-V3人类全基 因组芯片，对比调查了经多次照射产生放射抵抗性的食管癌细胞系 KYSE-170R 及其亲代细胞系 KYSE- 170 的基因表达谱差异。 由 qRT-PCR和 Western blot证实该基因确实存在差异表达。应用慢病 毒介导的 RNA干扰体系在体内外 差异基因与放射抵抗的关系， 并对该基因在食管癌患者病理组织中的表达情况进行回顾性分析。
试验结果表明食管癌细胞系 KYSE- 170R 及其亲代细胞系 KYSE- 170的基因在未照射、 照射后 8小时及照射后 24小时分别有 945， 733和 1232个表达差异基因。 qRT-PCR结果提示相对于亲代 细胞系 KYSE- 170， 放射抵抗细胞系 KYSE-170R在未照射、 照射后 8 小时及照射后 24小时 AKR1C3分别有 9. 39， 5. 94和 9. 20倍的表达 上调。 Western blot 结果证实， 在未照射的放射抵抗细胞系 KYSE-170R 中 AKR1C3 蛋白表达量显著高于亲代细胞系 KYSE- 170 ( P<0. 05 ) 。 应用慢病毒介导的 RNA干扰体系沉默 AKR1C3后， 放 射抵抗细胞系 KYSE- 170R的放射敏感性显著增强， 应用 AKR1C3过 表 粒转导亲代细胞系 KYSE- 170发现其放射抵抗性显著增强。 机制探讨初步结果提示， AKR1C3沉默引起的放射敏感性增强，很可 能与 R0S晚聚集和放疗后 48小时内 G2期细胞阻滞有关。
在对食管癌患者病理组织中的表达情况进行回顾性分析中，发 明人发现 AKR1C3表达于食管癌细胞， 正常食管粘膜细胞， 间质细 胞及小血管上皮中。正常食管粘膜细胞的表达量明显低于高分化食 管鳞癌细胞； 高分化食管鳞癌细胞表达量明显高于低分化食管鳞癌 细胞。 而且后续的研究表明， AKR1C3可能会为食管癌， 尤其是食管 高分化鳞癌， 提供新的放射增敏药物作用靶点。
总之， 经过上述多方面努力， 发明人终于找到了与食管癌抗性 相关的基因 - AKR1C3 ( Homo sapiens aldo-keto reductase family 1， member C3 (3— alpha hydroxysteroid dehydrogenase, type II) (AKR1C3) ,经检索发现该序列的 Genbank号为 NM-003739 ， 其全序 列如下：
1 gcccattgtt tttgtaatct ctgaggagaa gcagcagcaa acatttgcta gtcagacaag
61 tgacagggaa tggattccaa acaccagtgt gtaaagctaa atgatggcca cttcatgcct
121 gtattgggat ttggcaccta tgcacctcca gaggttccga gaagtaaagc tttggaggtc
181 acaaaattag caatagaagc tgggttccgc catatagatt ctgctcattt atacaataat
241 gaggagcagg ttggactggc catccgaagc aagattgcag atggcagtgt gaagagagaa
301 gacatattct acacttcaaa gctttggtcc acttttcatc gaccagagtt ggtccgacca
361 gccttggaaa actcactgaa gaaagctcaa ttggactatg ttgacctcta tcttattcat
421 tctccaatgt ctctaaagcc aggtgaggaa ctttcaccaa cagatgaaaa tggaaaagta
481 atatttgaca tagtggatct ctgtaccacc tgggaggcca tggagaagtg taaggatgca
541 ggattggcca agtccattgg ggtgtcaaac ttcaaccgca ggcagctgga gatgatcctc
601 aacaagccag gactcaagta caagcctgtc tgcaaccagg tagaatgtca tccgtatttc
661 aaccggagta aattgctaga tttctgcaag tcgaaagata ttgttctggt tgcctatagt
721 gctctgggat ctcaacgaga caaacgatgg gtggacccga actccccggt gctcttggag
781 gacccagtcc tttgtgcctt ggcaaaaaag cacaagcgaa ccccagccct gattgccctg
841 cgctaccagc tgcagcgtgg ggttgtggtc ctggccaaga gctacaatga gcagcgcatc 901 agacagaacg tgcaggtttt tgagttccag ttgactgcag aggacatgaa agccatagat 961 ggcctagaca gaaatctcca ctattttaac agtgatagtt ttgctagcca ccctaattat 1021 ccatattcag atgaatatta acatggaggg ctttgcctga tgtctaccag aagccctgtg 1081 tgtggatggt gacgcagagg acgtctctat gccggtgact ggacatatca cctctactta 1141 aatccgtcct gtttagcgac ttcagtcaac tacagctgag tccataggcc agaaagacaa 1201 taaattttta tcattttgaa ataa ( SEQ ID NO: 1 )
该序列可以参见 NCBI Genbank网页 http：〃 www. ncbi. nlm. nih. gov /nuccore/NM-003739. 4 ) ，
在通过多个试验确定 AKR1C3蛋白与放射抗性密切相关后， 发 明人对其进行了多方面检索， 发现醛酮还原酶超家族（AKR1C )与 肿瘤生物学非常密切。 AKR1C 家族为 37 kDa 可溶性蛋白， 可通过 NAD (P) (H)介导的氢离子传递作用， 将!^还原为羟基。 AKR1C3 属于醛酮还原酶超家族成员。 在人类， AKR1C 有四个亚型， 即 AKR1C1, AKR1C2, AKR1C3和 AKR1C4。 AKR1C3有大于 86 %的序列与 其余 3个 AKRs同源。 AKR1C3具有 3 oc -HSD， Π β -HSD, 20 -HSD, 和前列环素 （PG) F合成酶活性， 能够催化雄激素、 雌激素、 前列 腺素和前列环素及外源性物质的代谢。
AKR1C3 mRNA首先被发现与表达于乳腺和前列腺， 随后在肝脏、 肺、 小肠、 腎上腺、 脑、 子宫和睾丸陆续报道发现该基因的表达。
AKR1C3已被证明在多种类型癌症中表达失调，包括骨髄增生异常综 合征（MDS， 难治性贫血）， 子宫内膜癌，肺癌中表达上调。 但是目 前， AKR1C3在食管癌中的表达尚无报道。
为了构建药物合适的药物筛选模型，并且进一步确认放射抗性 与 AKR1C3的关系，发明人构建了系列靶向 AKR1C3的载体作为候选 药物在细胞水平和动物水平检测该筛选方法的可行性。具体是通过 所述构建的载体来沉默 AKR1C3的表达， 对沉默后的细胞进行放射 检验， 观察该基因沉默与放射抗性间的关系。
此处， 实施例中所用的候选药物是反义分子， 事实上还可以采 用各种已知的化合物库等对所述候选药物进行检测。也可以通过其 它能够对 AKR1C3的表达进 ^节的化合物例如蛋白核酸等进行筛 选。
具体地， 发明人建立了细胞筛选模型和动物筛选模型， 选取三 种已知对 AKR1C3基因有靶向作用的基因作为候选药物， 在动物体 上对 AKR1C3进行沉默筛选， 对沉默成功的裸鼠的放疗抗性进行检 验， 一方面验证了筛选模型的可靠性， 一方面对筛选到的目的序列 的增敏效果进行检验。
结果从三个候 ^^因中筛选到一个有效的沉 列，并且食管 癌荷瘤裸鼠试验结果表明， 沉默 AKR1C3 后， 放射抵抗细胞系 KYSE-170R的放射敏感性显著增强。 对放疗后残留的裸鼠肿瘤组织 进行 HE染色发现，沉默 AKR1C3后的放射抵抗细胞系 KYSE- 170R形 成的肿瘤组织内为大量瘢痕组织仅残留小量肿瘤细胞， 而对照的放 射抵抗细胞系 KYSE- 17 OR形成的肿瘤组织内有大量肿瘤细胞残留。 沉默 AKR1C3 将引起食管鳞癌细胞放射敏感性增强。 该结果将为 AKR1C3成为放疗增敏药物靶点提供生物学依据，尤其是针对食管高 分化鳞癌。 同时也 了本发明的增敏剂效果。
由此， 一方面， 本发明提供了用于食管癌放疗抗性的增敏治疗 的靶点 - AKR1C3基因，还提供了用于食管癌放疗抗性的增敏治疗的 基因药物， 其为 AKR1C3基因表达抑制剂， 或者沉默剂， 优选地， 所述增敏剂是反义核酸。 任选地， 其中含有药学上可接受的佐剂。
另外一方面，本发明提供了用于食管癌放疗抗性的增敏治疗的 方法， 其包括使 AKR1C3基因沉默或者表达抑制的步骤。 优选地， 在所述步骤之前， 对待治疗个体进行食管癌细胞分类检验， 优选对 食管鳞癌个体实施所述步骤，进一步优选对食管鳞癌高分化个体实 施所述步骤。 具体地， 所述步骤为对有此需要的个体给予有效量的 AKR1C3基因沉默剂或者 AKR1C3基因抑制剂。
一方面，本发明提供了一种非治疗目的用于食管癌放疗抗性细 胞的筛选， 其包括对所述细胞实施 AKR1C3基因沉默或者表达抑制 的步骤。 其中所述细胞是离体的， 或者所述细胞是来自食管鳞癌个 体的， 优选来自食管鳞癌高分化个体的。
另外一方面，本发明提供了一种非治疗目的用于食管癌放疗抗 性动物的筛选， 其包括对所述动物实施 AKR1C3基因沉默或者表达 抑制的步骤。 所述动物可以是荷有食管癌放疗抗性细胞的大鼠， 小 鼠或者兔子等。 本发明中使用 AKR1C3基因沉默或者表达抑制的步 骤包括施用药物实现这一效果，所述的药物，例如为基因治疗药物。 也可以通过基因敲除技术等使之沉默。 一方面， 本发明提供了 AKR1C3基因表达抑制剂或沉默剂在制 备用于食管癌放疗抗性的增敏治疗的基因药物中的用途， 其中， 所 述的抑制剂可以是茉莉酸甲酯 （Methyl jesmonate, MeJ) ; 茉莉酸 (Jesmonate, J A)。
在本发明中，所述的沉默剂是指能够在体外或者体内使 AKR1C3 基因不表达的化合物， 其可以是核酸片段， 蛋白质， 糖类等， 也可 以是化学小分子。
本发明中， 所述的抑制剂是指能够在体外或者体内使 AKR1C3 基因不表达或者低表达的化合物， 其可以是核酸片段， 蛋白质， 糖 类等， 也可以是化学小分子。 其作用的靶点可以是 AKR1C3基因本 身， 也可以是 AKR1C3基因表达的调控元件， 例如启动子等。
进一步地， 一方面， 本发明提供了食管癌放射抗性增敏剂的初 步筛选方法， 其包括：
( 1 )提供高表达 AKR1C3基因的宿主细胞；
( 2 )提供候选药物；
( 3 )使（ 1 ) 的宿主细胞和 ( 2 ) 的候选药物在合适的条件下 接触；
( 4 )检测 （3 ) 中的经过候选药物作用的宿主细胞的 AKR1C3 基因的表达；
能够使 AKR1C3基因表达下调或者消失的候选药物， 判定为具 有食管癌放射抗性增敏剂潜力的药物。
任选地， 可以进行随后的动物试验等进行验证。
其中， 高表达 AKR1C3基因的宿主细胞是指相对未转染 AKR1C3 基因的宿主细胞其表达量高。 另外一方面，本发明提供了食管癌放射抗性增敏剂的细胞水平 筛选方法， 其包括
(1)提供高表达 AKR1C3基因的食管癌细胞；
(2)提供候选药物；
( 3 )使（ 1 )的食管癌细胞和（ 2 )的候选药物在合适的条件下接 触； （ 4 )检测（ 3 )中的经过候选药物作用的食管癌细胞的 AKR1C3 基因的表达；
(5)能够使 AKR1C3基因表达下调或者消失， 同时保持食管癌细 胞存活的候选药物可以判定为是食管癌放射抗性增敏剂。
其中， 高表达 AKR1C3基因的食管癌细胞是指相对放射敏感的 食管癌细胞其表达量高， 例如 KYSE- 170R细胞系。
其中， 所 i½合适的 下接触， 是指使高表达 AKR1C3基因 的食管癌细胞的正常生长除了受候选药物影响外，其它生长条件与 常规生长条件一致。 一方面，本发明还提供了食管癌放射抗性增敏剂的动物水平的 筛选方法， 其包括
(1)提供荷有高表达 AKR1C3基因的食管癌肿瘤的动物；
(2)提供候选药物；
(3)将足够量的 （2)的候选药物给予 （1)的动物， 连续给药一 定时间； ( 4 )检测 ( 3 ) 中的给药后的动物所荷肿瘤的大小；
( 5 )能够使 AKR1C3基因表达下调或者消失， 同时使肿瘤变小或者 消失的的候选药物可以判定为是食管癌放射抗性增敏药物。
其中， 高表达 AKR1C3基因的食管癌细胞是指相对放射抗性的 食管癌细胞其表达量高， 例如 KYSE- 170R细胞系。 其中所述动物可 以是裸鼠， 也可以是其它试验动物。
其中的给予方式可以是通过口腔给药， 静脉输注， 或者瘤内注 射等。 以下， 通过附图对本发明进行说明。
附图说明
图 1:表示的是放射抵抗细胞系 KYSE- 170R和非放射抗性细胞系 KYSE- 170间的差异表达基因的比较结果。 其中，
图 I-A: KYSE-170R及 KYSE-170细胞系芯片差异比较结果。 可 以发现，在未照射、照射后 8小时及照射后 24小时分别有 945， 733 和 1232个表达差异基因。 其中， AKR1C3在 3个时间点的 I l lumine 差异值均 > 50。其中 I l lumine差异值 > 20被认为 ½因表达上调； I l lumine差异值 < -20被认为 因表达上调。
图 1- B:应用 qRT- PCR在 mRNA 7j平上的验证，由图可发现在 mRNA 水平 AKR1C3表达差异显著。 与亲代 KYSE- 170细^目比， 在照射后 Oh, 8h及 24h， 抵抗的 KYSE- 170R细胞中 AKR1C3的表达分别上调 了 9. 39， 5. 94和 9. 20倍。
图 1- C: Western blot结果， 该结果证实， 在未照射的放射抵 抗细胞系 KYSE- 170R 中 AKR1C3 蛋白表达量显著高于亲代细胞系 KYSE- 170 ( *P<0. 05 ) 。
图 2: AKR1C3沉默的 KYSE- 170R 细胞和亲代 KYSE- 170细胞中 过表达 AKR1C3后的放射抗性检测结果。 其中
图 2- A: 表示 qRT- PCR的结果，其表明 AKR1C3沉默效率达 60%。 图 2- B、 C: 表示 western blot 的结果， 其证实在蛋白水平， AKR1C3表达明显下调。
图 2- D、 E: AKR1C3沉默的 KYSE- 170R 细胞的放射抗性检测， 克隆形成试验结果提示， 未照射时， scramble- shRNA-KYSE-170R 及 AKR1C3- shRNA- KYSE-170R 细胞克隆形成率无显著性差异， 说 明 AKR1C3沉默并不影响 KYSE- 170R的增殖。 经 2， 4， 8Gy照射后， AKR1C3沉默的 KYSE- 170R 细胞克隆形成数显著减少， 放射敏感性 明显增强。 E 中國代表 scramble- shRNA- KYSE- 170R细胞组； *代 表 AKR1C3- shRNA- KYSE-170R细胞组。
图 2- F、 G: 在对放射相对敏感的亲代 KYSE- 170细胞中过表达 AKR1C3后的 Western blot验证结果。 其中将 AKR1C3过表达质粒 瞬时转染亲代细胞株 KYSE- 170，并瞬时转染 scramble质粒作为对 照。 结果 Western blot验证过表达效果显著。
图 2- H、 I： 克隆形成试验检测 AKR1C3过表达的 KYSE- 170 细 胞的放射抗性。 结果未照射时 scramble- KYSE- 170 及 AKR1C3+ KYSE- 170 细胞克隆形成率无显著性差异，说明 AKR1C3过表达并不 影响 KYSE- 170 的增殖。 经 2， 4， 8Gy照射后， AKR1C3过表达的 KYSE- 170 细胞克隆形成数显著增多，放射抵抗性明显增强。 I中國 代表 AKR1C3+ KYSE- 170细胞组； *代表 scramble- KYSE- 170细胞 组。
图 3: 裸鼠成瘤模型检测靶基因沉；1½对瘤体放射抵抗性的影 响。
图 3-A: 当瘤体直径达到 6-8隨时， 第 2组和第 4组给予 15Gy 的照射剂量单次局部照射的状况。
图 3- B: 根据观察结果， 绘制裸鼠肿瘤体积变化曲线。 曲线提 示在接受放疗后， 与接种 scramble- shRNA- KYSE- 170R细胞相比，稳 定沉默 AKR1C3的 AKR1C3- shRNA- KYSE- 170R细胞所形成的肿瘤， 体 积缩小更为显著； 而两组在未接受放疗时， 肿瘤生长速度无明显差 异。 *代表 s cr amb 1 e-shRNA-KYSE-17 OR 细胞组； 國代表 AKR1C3- shRNA- KYSE- 170R 细 胞 组 ； ▲ 代 表 s cr amb 1 e-s hRNA-KYSE-17 OR 细 胞 + 放 射 治 疗 组 ； + AKR1C3- shRNA- KYSE- 170R细胞 +放射治疗组。
图 3- C: 对未接受放疗的裸鼠肿瘤组织进行免疫组织化学法检 测其中的 AKR1C3蛋白的结果。 其为 scramble- shRNA-KYSE-170R组 AKR1C3蛋白表达量明显高于 AKR1C3- shRNA- KYSE- 170R组。
图 3-D: 对未接受放疗的裸鼠肿瘤组织进行免疫组织化学法检 测其中的 AKR1C3蛋白的结果， 其为 AKR1C3- shRNA- KYSE- 170R组。
图 3- F: 对放疗后残留的裸鼠肿瘤组织进行 HE染色结果， 其表 明 AKR1C3- shRNA- KYSE- 170R 细胞所形成的肿瘤组织内为大量瘢痕 组织仅残留小量肿瘤细胞。
图 3- E: 对放疗后残留的裸鼠肿瘤组织进行 HE染色结果， 其表 明作为对照的 scramble- KYSE-170R 细胞形成的肿瘤组织内有大量 肿瘤细胞残留。
图 3-G: 取第 1组 scramble-KYSE-170R组及第 3组 AKR1C3- shRNA— KYSE— 170R组各 3只进行 qRT— RCR的结果。 图 3— H， I： 取第 1 组 scramble- KYSE-170R组及第 3组 AKR1C3- shRNA- KYSE- 170R组各 3只进行 western blot的结果。
图 4:在应用免疫组织化学法，对食管癌患者病理组织中 AKR1C3 的表达情况进行回顾性分析结果。 其中
图 4- A: 在 6例食管低分化鳞癌患者病理标本中的表达， 图 4- B: 在 6例食管高分化鳞癌患者病理标本中的表达； 图 4- C: 在癌旁正常食管粘膜细胞病理标本中的表达。
图 4- D: 图 4A- C的结果比较图。
以下， 通过具体实施例对本发明进行说明， 此处所述的实施例 仅用来阐述本发明， 其不用于限定本发明的保护范围， 任何本领域 技术人员根据现有技术和本发明的教导得到的本发明的变体或者 等价的发明都包含在本发明的保护范围内。
具体实施方式
实施例 1: 放疗抗性基因的确认
1. 细胞系和放疗方法：
人类食管鳞癌细胞系 KYSE—170 (保藏曰 2011年 6月 14曰，保藏 号 CGMCC No: 4937， 分类命名为人类食管鳞癌细胞系的 KYSE - 170， 保藏地： 中国 微生物菌种保藏管理中心 菌种保藏地址： 地址： 北京市朝阳区北辰西路 1 号院， 中国科学院 ^物研究所），及经过 照射后形成的放射抵抗细胞系 KYSE170R (保藏地： 中国普通 ^ 物菌种保藏管理中心 菌种保藏地址： 地址： 北京市朝阳区北辰西路 1 号院， 中国科学院 ^物研究所，保藏曰 2011年 6月 14 曰， 保藏号 CGMCC No: 4936， 分类命名为人类食管鳞癌细胞系的 KYSE - 17 OR )。 以上两株细胞均由美国 MD Anderson Cancer Center胸部肿瘤放射治 疗科惠赠。以上细胞系应用高糖 1640培养液 (购自 Invitrogen公司）， HEK293T 细胞（由北京大学药学院国家重点实验室提供)培养于高糖 DMEM培养液（购自 Invitrogen公司）。 上^养液临用时加入 10% 的灭活胎牛血清 (FBS,购自 HyClone公司 )及青链霉素 (各 50 -^/mL) 即可。 培养条件： 37 温箱， 5% C02。 应用 Backman Z1细胞计数仪计 数细胞个数（购自美国 Backman公司 )。 于北京大学第一临床医院肿 瘤放射治疗科， 应用 6MV- XH|†线， 对细胞进行照射（直线加速器购自 美国瓦里安公司）。
2. 细胞总 RNA的提取和基因芯片结果
给予处于对数生长期的 KYSE- 170及 KYSE- 170R细胞 4Gy剂量 照射， 应用 Total RNA提取试剂盒 ( SV Iosolation System购自 promega公司）， 在照射前（Oh )及照射后不同时间点（ 8h， 24h ) 提取细胞总 RNA。 应用 I l lumine- 6-V3人类全基因组芯片， 筛选 KYSE- 170及 KYSE- 170R在上述时间点基因的表达变化差异（由上海 生物芯片有限公司完成） 。 结果参见图 1， 其中 Illumine差异值 > 20被认为 因表达 上调； Illumine差异值 < -20被认为 因表达上调。 对比分析 KYSE- 170R及 KYSE- 170细胞系芯片结果发现， 在未照射、 照射后 8 小时及照射后 24小时分别有 945， 733和 1232个表达差异基因。 其中， AKR1C3在 3个时间点的 Illumine差异值均>50， 所以发明 人选取该基因进行下一步实验。
3. 应用 qRT-PCR及 Western blot验证芯片结果
( 1 )给处于对数生长期的 KYSE- 170及 KYSE- 170R细胞予 4Gy 剂量照射， 应用 Total RNA提取试剂盒（ SV losolation System 购自 promega公司），在照射前（Oh)及照射后不同时间点 （ 8h， 24h)H取细胞总 RNA。应用逆转录试剂盒 (AMV Reverse Traslation System购自 promega公司）将总 RNA逆转录成 CDNA， 应用 Go Taq qPCR Master Mix 酵（购自 promega公司）行 qRT—PCR。
PCR引物由 Primer3.0软件设计， 并经 Blast 其特异性。 目标序列： AKR1C3 (GenBank号： M-003739, SEQ ID NO: 1 ) 上游引物： 5， atttggcacctatgcacctc 3， (SEQ ID NO: 2);
下游引物 5， tgagttttccaaggctggtc 3， (SEQ ID NO: 3);
目标序列： β -acting
上游引物： 5， agcgagcatcccccaaagtt 3， (SEQ ID NO: 4) ； 下游引物： 5， gggcacgaaggctcatcatt 3， (SEQ ID NO: 5)。 应用 Roche 480 PCR仪（美国 Roche公司）行 PCR反应， 具 体条件： 第一个循环（95 - 10分钟）； 第二个循环（95 - 30秒， 60 - 30秒， 72 - 30秒） x40。 mRNA表达差异倍数计算公式如下： ACT (KYSE170R/KYSE170) = CT(AKR1C3) - CT ( β -acting)； ΔΔ CT=ACT (KYSE-170R) - ACT (KYSE— 170)； 表达差异倍数 =2~ C 应用 qRT- PCR验证后发现在 mRNA水平 AKR1C3表达差异显著。 与亲代 KYSE- 170 细胞相比， 在照射后 0h， 8h及 24h， 抵抗的 KYSE-170R细胞中 AKR1C3的表达分别上调了 9. 39， 5. 94和 9. 20 倍 (见图 1-B)。
( 2 )应用 Western blot分析 AKR1C3KYSE- 170及 KYSE-170R 细胞中的含量。
取对数生长期的 KYSE- 170及 KYSE- 170R细胞， 用冷的 PBS 洗 2 次， 根据细胞量的多少， 加入适量蛋白裂解液 （ RPIA 购自 Applygene公司） ， 置于冰浴上裂解 30min。 4*Ό 14000 rpm 离心 30min， 弃下层沉淀， 收集上清即为细胞全蛋白。 应用 Bio- Rad蛋 白定量试剂盒 （Bio- Rad, Hertfordshire, United Kingdom)定量 蛋白， 分装。 应用 12% SDS-聚丙烯酰胺凝胶电泳分离蛋白， 将凝胶 上蛋白转移至 ECL Chemi luminescent substrate 膜 (Amersham Pharmacia Biotech, Piscataway, NJ, USA上， 用含有 5%脱脂奶 粉和 0. 1%吐温的封闭液室温封闭 1小时。 一抗 孵育过夜， 次曰 用 TBST洗 4次， 每次 5min， 然后于二抗中室温孵育 2小时。 TBST 洗 4次，每次 15 min。所用抗体鼠抗人 AKR1C3单克隆抗体（ 1： 1000 ) 购自 Sigma公司； 兔抗人 GAPDH单克隆抗体（1: 1000 )购自 Santa Cruza公司。 二抗为辣根过氧化物酶标记的羊抗鼠及羊抗兔单克隆 抗体（1: 4000) 购自 Santa Cruza公司。 所有 Western blot至少重 复 2次， 实验结果应用 Quantity One imaging program分析程序 进行相对定量分析 （Bio- Rad, Hercules, CA)。
上述 Western blot 结果证实， 在未照射的放射抵抗细胞系 KYSE-170R 中 AKR1C3 蛋白表达量显著高于亲代细胞系 KYSE- 170 ( *P<0. 05 ) , 见图 1-C 。 实施例 2: 用放疗抗性细胞建立药物筛选模型
1. 沉默 AKR1C3的 shRNA慢病毒质粒载体构建和最佳 shRNA载体筛 选 为了构建药物合适的药物筛选模型，并且进一步确认放射抗性 与 AKR1C3的关系， 发明人选择放疗抗性 KYSE- 170R细胞系作为筛 选细胞， 同时构建了系列靶向 AKR1C3的载体作为候选药物。 具体 是通过所述构建的载体来沉默 AKR1C3的表达， 对沉默后的细胞进 行放射检验， 观察该基因沉默与放射抗性间的关系。
慢病毒（ Lent i virus )载体是以 HIV- 1 (人类免疫缺陷 I型病 毒）为基础发 ½来的基因治疗载体。区别一般的逆转录病毒载体， 它对分裂细胞和非分裂细胞均具有感染能力。该载体可以将外源基 因有效地整合到宿主染色体上， 从而达到持久性表达。 发明人应用 优化改建的慢病毒载体 PSD31 ( Zhang J等人， A more eff icient RNAi inducible system for tight regulation of gene expression in mammal ian cells and xenograft animals. RNA. 2007 Aug; 13 (8) : 1375-83. )， 携带可以靶向沉默 AKR1C3的 shRNA序列， 转染细胞， 沉默目的基因。
发明人查阅 Sigma公司网络数据库，选取 3组靶向沉默 AKR1C3 基因的 shRNA序列进行载体构建， 分别命名为 shRNA ( 0 ) ； shRNA ( 1 )； shRNA ( 2 )其引物序列和具体靶点见下。 shRNA引物由北京 华大基因合成。 应用人类 U6启动子， 启动转录过程。
其中， 人类 U6 启动子序列如下：
5' -TGGATCCAAGGTCGGGCAGGAAGAG-3' (SBQ ID NO: 6)，
作为阴性对照的无功能 siRNA (scramble) ， 上， 下游引物序 歹1 J见文献 ( Zhang J等人， A more eff icient RNAi inducible system for tight regulation of gene expression in mammal ian cells and xenograft animals. RNA. 2007 Aug; 13 (8) : 1375-83 ) ，
其中， scramble的下游引物序列如下：
下游引物：
5' -CTCTATCATTGATAGAGTGACTCCAGTGGTAATCTACCTCTTCTTTACCTTCTTTA-3' (SBQ ID NO: 7)，
3条沉默 AKR1C3的序列的下游引物序列如下： shRNA (0) : 其对应于 SEQ ID NO: 1的碱基位置： 148- 168
5' -AGGATCCAAAAACCAGAGGTTCCGAGAAGTAAACTCGAGTTTACT TCTCGGAACCTCTGGCCGGTGTTTCGTCCTTTCCAC-3' (SEQ ID NO: 8)；
shRNA (1) 其对应于 SEQ ID NO: 1的 位置 693-983:
5' -AGGATCCAAAAACCTAGACAGAAATCTCCACTACTCGAGTAGTGGAG ATTTCTGTCTAGGCCGGTGTTTCGTCCTTTCCAC-3' (SEQ ID NO: 9)；
shRNA (2) 其对应于 SEQ ID NO: 1的碱基位置 372-390:
GTTTCGTCCTTTCCAC-3' (SEQ ID NO: 10) .
shRNA (0) - ( 2 )这三个沉默载体的沉; I ^列的上游引物均为:
5' -TGGATCCAAGGTCGGGCAGGAAGAG-3' (SEQ ID NO: 11) . hU6-shRNA 序列通过 PCR 方法合成， 具体步骤参见文献 [Y. Chen 等人， Down-regulation of CXCR4 by inducible smal l interfering RNA inhibits breast cancer cel l invasion in vitro. Cancer Res. 2003 (63) 4801 - 4804 ; S. Matsukura 等人， Establ ishment of conditional vectors for hairpin si RNA knockdowns, Nucleic Acids Res. 31 (2003) e77]。
应用 T4 DNA 连接试剂盒 （NEB, Beverly, MA, USA) , 将上述 PCR产物与 pGEM T easy vector载体进行连接， 送华;^因公司进 行 DNA测序验证连接正确性。 测序正确的 TA克隆质粒， 根据 mega tans 1. 0 (0rigen)转染试剂盒步骤， 瞬时转染 KYSE- 170R细胞。 然 后进行 Western blotting进行选择。
结果发现通过 Western blotting实验验证， 在候选的三个针 对 AKR1C3的沉; I ^因中， 载体 shRNA ( 2 ) 的效果最好。 即反义序 列 SEQ ID NO: 1的碱基位置 372-390能够有效沉默 AKR1C3的表达。 在用 pGEM T easy vector载体进行瞬时转染验证成功后， 发 明人为了建立稳定的筛选模型， 又构建了可以稳定转染的载体。 具体方法应用 BamHI FastDigest内切酵（购自 Fermentas公 司）酶切前述 的沉默效果好的 AKR1C3 -shRNA ( 2 ) , 并选择了 PSD31慢病毒载体（携带 puromycin抗性基因 )做为新的载体， 将 该载体进行酶切，制造连接粘末端，通过 Rapid DNA Dephos & Ligion Ki t连接试剂盒 (ROCHE)将两者连接， 测序验证连接成功。
2. 构建能够稳定沉默靶基因 AKR1C3的载体
应 用 mega tans 1. 0 (Origen) 转 染 试 剂 盒 ， 将 PSD31- AKR1C3- shRNA及病毒包装辅助质粒 VSVG、 PRSV及 PMDL质 粒（购自 Invi trogen公司 )共转染 HEK 293T细胞（购自 Invitrogen 公司）， 进行病毒包装， 应用 mega tans 1. O (Origen)转染试剂盒。
其中， 共转染 4 个质粒就是病毒包装过程。 其中， PSD31- AKR1C3- shRNA质粒是形成病毒的核心部分， VSVG、 PRSV及 PMDL病毒包装辅助质粒是辅助核心质粒， 起作用是辅助病毒包膜形 成， 细胞吐病毒等过程的。
收集 48 小时及 72 小时上清（病毒液）转导 KYSE- 170R 经 Puromycin筛选 10 应用 qRT- PCR和 Western blot ^ £。 qRT- PCR结果表明， AKR1C3沉默效率达 60% (图 2- A )， western blot 结 果证实在蛋白水平， AKR1C3表达明显下调（ *P<0. 05 ) (参见图 2-B、 C )。
以上结果表明发明人建立了稳定表达 PSD31- AKR1C3- shRNA的 KYSE-170R细胞系。
由此，发明人通过有效的细胞模型筛选得到了有效的 AKR1C3表 达沉默剂。
3.克隆形成实验对比观察靶基因沉; 1½细胞系与原抵抗细胞系放射 敏感性的变化
克隆形成实验是评价放射治疗效果的经典实验。发明人将检测在 不同照射剂量下， AKR1C3基因稳定沉默细胞系克隆形成数量与未沉 默细胞系 scramble-shRNA-KYSE-170R (该株系是与 AKRlC3-shRNA -KYSE-170R 同时建立的作为阴性对照的细胞株。 不同之处是， scramble-KYSE-170R连入的 A t功能基因 scramble。具体见建立稳 定沉默 AKR1C3细胞系试验）的区别。克隆形成数量反映了该细胞系 对放射治疗的敏感性。 克隆形成越多， 其敏感性越低， 抵抗性越强。
给 予 对 数 生 长 期 的 scramble- KYSE-170R 及 AKRlC3-shRNA-KYSE-170R 细胞照射， 剂量依次为 （2， 4， 8 Gy)， 然后胰酶消化， 细胞计数仪 Vi-CELL (购自 BECKMAN公司）计数 细胞。 根据不同照射计量， 调整接种细胞数量。
在相同剂量下， 将等量的 scramble-KYSE-170R及 AKRlC3-shRNA -KYSE-170R细胞接种于六 上， 经 7-10天培养， 取出， 固定， 结晶紫染色 （Sigma Chemical Co. ) , 应用计数克隆数（包含 50个 及以上细胞的记为一个克隆）。计数相应细胞未照射时的克隆数以纠 正接种率， 增强可比性。
克隆形成试验结果示于表 1， 该结果提示， 未照射时， scramble- shRNA-KYSE-170R 及 AKR1C3- shRNA- KYSE- 170R 细胞克 隆形成率无显著性差异， 说明 AKR1C3沉默并不影响 KYSE- 170R的 增殖。 经 2， 4， 8Gy照射后， AKR1C3沉默的 KYSE- 170R 细胞克隆 形成数显著减少， 放射敏感性明显增强 （*Ρ<0· 05， ** Ρ<0. 01 ) 。
(见图 2- D、 E) 表 -1 AKR1C3沉默的 KYSE-170R 细胞克隆形成率变化表 (均数 ±标准差）
scramble-s hRNA-IYSE-17 OR AlRlC3-shRNA-IYSE-170R
0GY 1 1
2GY 0. 6646 ± 0. 2178 0. 3740 ± 0. 0873
4GY 0. 2267 ± 0· 0236 0. 0433 ± 0. 135
8GY 0. 0190 ± 0. 055 0. 0014 ± 0. 0005 4. 过表达 AKR1C3的放疗敏感细胞建立的药物筛选模型 既然 AKR1C3沉默后， 放射抵抗的 KYSE- 170R 细胞放射敏感性 增强， 发明人将 AKR1C3 过表达质粒 （Origene 公司 Cat. No: SC321532 ) 瞬时转染亲代细胞株 KYSE- 170，方法同上。 瞬时转染 scramble质粒作为对照。 对放射相对敏感的亲代 KYSE- 170细胞中 过表达 AKR1C3,探索其放射敏感性的变化和药物筛选效果。 Western blot ^it^Ji^^^著（ *Ρ<0· 05 ) (图 2—F、 G )。
克隆形成试验结果示于表 2， 该结果提示， 未照射时 scramble- KYSE- 170及 AKR1C3+ KYSE- 170 细胞克隆形成率无显著 性差异， 说明 AKR1C3过表达并不影响 KYSE- 170的增殖。 经 2， 4， 8Gy照射后， AKR1C3过表达的 KYSE- 170 细胞克隆形成数显著增多， 放射抵抗性明显增强 （ *Ρ<0· 05 ， ** Ρ<0. 01 ) 。 （见图 2-Η、 I) 表- 2 AKR1C3过表达的 KYSE- 170 细胞克隆形成率变化表（均 数士标准差）
scramble-KYSE-170 AKR1C3+ KYSE-170
0GY 1 1
2GY 0. 564 ± 0. 1409 0. 8586 ± 0. 2185
4GY 0. 3150 ± 0. 0296 0. 5767 ± 0. 0785
8GY 0. 0070 ± 0. 0019 0. 0580 ± 0. 0172 在上述实施例 1中，发明人选择遗传背景一致的食管鳞癌抵抗 细胞株 ( KYSE-170R )及亲代细胞株（KYSE- 170 )未照射及照射后 不同时间点的基因表达傳差异， 动态分析照射前后基因的表达变 化， 在一定程度上避免了因接受照射启动放射抵抗基因表达， 而导 致的漏选情况。根据倍数差异、 GO分析和 Pathway分析， 推测放射 抵抗细胞中表 著上调的 AKR1C3等 10个基因可能参与了放射抵 抗的形成。进一步的体外验证结果表明， AKR1C3基因与食管癌放射 抵抗相关。
各个试验的综合结果可以确定 AKR1C3 基因是放射抗性细胞 KYSE-170R和放射敏感细胞 KYSE-170的表达差异基因，在放射抗性 细胞 KYSE- 170R中沉默 AKR1C3基因导致细胞的放射抗性降低， 在 放射敏感细胞 KYSE- 170 i± 达 AKR1C3基因能够提高该细胞的放射 抗性， 证明 AKR1C3基因是与放射抗性相关的重要基因。 实施例 3: 动物筛选模型的建立和基因沉默方法的增敏效^ r验
应用 4-6周龄的雄性裸鼠，随机分为 4组（每组 5只）：第 1组： scramble- KYSE- 170R组； 第 2组： scramble- KYSE- 170R+放疗组； 第 3 组 ： AKR1C3- shRNA- KYSE- 170R 组 ； 第 4 组 ： AKR1C3- shRNA- KYSE- 170R+放疗组。将处于对数生长期的人类食管癌 细胞 scramble- KYSE- 170R及 AKRlC3-shRNA- KYSE- 170R胰酶消化， PBS 洗涤， 重悬成浓度为 2 x l07l00ul PBS。 将上述两种细胞悬液 100 ul 分别接种于裸鼠后腿皮下。 当瘤体直径达到 6-8随时， 第 2 组和第 4组给予 15Gy的照射剂量单次局部照射。照射后观察肿瘤体 积变化。 肿瘤体积计算公式 V = π / 6 (a X b2)， a为长径， b为短径。 连续观察 36天， 或观察到肿瘤体积达到 1. 0 cm3。 具体结果见表 3， 试验过程见图 3-A。 表- 3裸鼠肿瘤体积 -天数变化表 (均数士标准差）
Figure imgf000021_0001
根据观察结果， 绘制裸鼠肿瘤体积变化曲线（见图 3- B )。 曲线 提示在接受放疗后， 与接种 scramble- KYSE-170R细^目比，稳定沉 默 AKR1C3的 AKR1C3- shRNA- KYSE- 170R细胞所形成的肿瘤， 体积缩 小更为显著； 而两组在未接受放疗时， 肿瘤生长速度无明显差异。
对未接受放疗的裸鼠肿瘤组织进行免疫组织化学法， 检测其中 的 AKR1C3蛋白，结果提示 scramble- KYSE-170R组（见图 3- C )AKR1C3 的蛋白表达量明显高于 AKR1C3- shRNA- KYSE- 170R组（见图 3-D )。
对放疗后残留的裸鼠肿瘤组织进行 HE 染色发现， AKRlC3-shRNA-KYSE-170R细胞所形成的肿瘤组织内为大量瘢 织 仅残留小量肿瘤细胞（见图 3- F )， 而对照的 scramble- KYSE-170R 细胞形成的肿瘤组织内有大量肿瘤细胞残留 （见图 3-E )。
取 第 1 组 scramble- KYSE-170R 组 及 第 3 组 AKR1C3- shRNA- KYSE- 170R组各 3 只进行 qRT-RCR (见图 3- G )及 western blot (见图 3- H， I )试验， 进一步 两组中 AKR1C3的表 达差异。 结果提示 AKR1C3在 scramble- KYSE-170R组表达确实高于 AKR1C3- shRNA- KYSE- 170R组（ *Ρ<0· 05 )。
由此， 证明所述的动物模型可以用于放疗抗性增敏剂的筛选。 实施例 4: 统计学方法回顾性分析食管癌患者病理组织中 AKR1C3的 表达情况
在应用免疫组织化学法， 对食管癌患者病理组织中 AKR1C3的 表达情况进行回顾性分析中， 发明人发现其表达于食管癌细胞（见 图 4- Α，Β ) ， 正常食管粘膜细胞（见图 4- C ) 中。 课题组选取 6例 食管高分化鳞癌患者病理标本（见图 4- Β )； 6例食管低分化鳞癌 患者病理标本（见图 4- Α )及癌旁正常食管粘膜细胞病理标本（见 图 4- C )进行回顾性分析。 正常食管粘膜细胞的表达量明显低于高 分化食管鳞癌细胞； 高分化食管鳞癌细胞表达量明显高于低分化食 管鳞癌细胞（ **Ρ<0· 01 ) (见图 4- D,其中 1为低分化食管鳞癌细胞 组； 2为高分化食管鳞癌细胞组； 3为正常食管粘膜细胞组） 。 在食管癌临床治疗中， 高分化食管鳞癌患者肿瘤的放射敏感性 低于低分化食管鳞癌患者已成为共识。 目前普遍认为， 高分化的食 管鳞癌预后较好， 但是与中低分化鳞癌相比， 其放射敏感性差。 人 食管鳞癌组织切片免疫组化的结果表明， 在正常食管粘膜、 间质和 小血管中有 AKR1C3的表达。 与低分化的食管鳞癌组织及正常食管 粘膜相比， AKR1C3在高分化的食管鳞癌组织中表达显著增高，进一 步说明 AKR1C3的高表达与食管鳞癌的放射抵抗有关。
在统计分析中， Student t- Test 用于比较两组间均数差异， 应用 SPSS 10. 0进行统计学分析。 P<0. 05被认为有统计学差异。

Claims

1. 逆转或降低食管癌放疗抗性的增敏药物，其为 AKR1C3基因表达 抑制剂或沉默剂。
2. 权利要求 1的增敏药物， 其中还含有药学上可接受的佐剂。
3. 权利要求 1的增敏药物， 其中的食管癌是食管鳞癌。
4. AKR1C3基因表达抑制剂或沉默剂在制备用于逆转或降低食管癌 放疗抗性的增敏治疗的药物中的用途。
5. 食管癌放射抗性增敏剂的初步筛选方法， 其包括
( 1 ) 提供高表达 AKR1C3基因的宿主细胞；
( 2 ) 提 ^选药物；
( 3 ) 使（ 1 ) 的宿主细胞和（ 2 ) 的候选药物在合适的条件下接 触；
( 4 ) 检测 （ 3 ) 中的经过候选药物作用的宿主细胞的 AKR1C3基 因的表达； 和
( 5 ) 能够使 AKR1C3基因表达下调或者消失的候选药物，判定为 具有食管癌放射抗性增敏剂潜力的药物。
6. 食管癌放射抗性增敏剂的细胞水平筛选方法， 其包括
( 1 ) 提供高表达 AKR1C3基因的食管癌细胞；
( 2 ) 提 ^选药物；
( 3 ) 使（ 1 ) 的食管癌细胞和（ 2 ) 的候选药物在合适的条件下 接触；
( 4 ) 检测 （3 ) 中的经过候选药物作用的食管癌细胞的 AKR1C3 基因的表达；
( 5 ) 将使 AKR1C3基因表达下调或者消失，同时保持食管癌细胞 存活的候选药物判定为食管癌放射抗性增敏剂。
7. 权利要求 6的方法， 其中的食管癌细胞是 KYSE- 170R细胞系或 者转化了 AKR1C3表达载体的 KYSE-170细胞系。
8. 食管癌放射抗性增敏剂的动物水平的筛选方法， 其包括
( 1 ) 提供荷有高表达 AKR1C3基因的食管癌的动物；
( 2 ) 提 ^选药物；
( 3 ) 将足够量的 （ 2 ) 的候选药物给予 （ 1 ) 的动物， 连续给药 一定时间；
( 4 ) 检测（3 )中的给药后的动物所荷肿瘤的大小或所荷肿瘤中 的 AKR1C3基因表达水平；
( 5 )使 AKR1C3基因表达下调或者消失， 同时使肿瘤变小或消失的 候选药物判定为食管癌放射抗性增敏潜在药物。
9. 一种非治疗目的体外逆转或降低食管癌细胞放疗抗性的方法， 其包括对实施沉默或者表达抑制 AKR1C3基因的步骤。
10. AKR1C3基因检测剂在制备用于检测患者食管癌放疗抗性高低、 放疗敏感性高低或者区分鳞癌分化程度的药物中用途。
11. 权利要求 10的用途， 其中的 AKR1C3基因检测剂是特异性检测 AKR1C3基因的 DNA探针。
12. 权利要求 11的用途， 其中特异性检测 AKR1C3基因的 DNA探针 固定在载体上。
13. AKR1C3基因在筛选用于逆转或降低食管癌放疗抗性的增敏治疗 的药物中的用途。
14. AKR1C3基因作为食管癌病人不适合放疗或放疗效果不显著的诊 断生物标志物的用途。
15. AKR1C3基因作为食管癌病人对放疗的敏感性差的诊断生物标志 物的用途。
16. AKR1C3作为区分食管高分化鳞癌和低分化鳞癌的生物学标志物 的用途。
PCT/CN2012/077434 2011-06-23 2012-06-25 逆转或降低食管癌放疗抗性的增敏剂、筛选方法及其用途 Ceased WO2012175052A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110169897.7 2011-06-23
CN2011101698977A CN102836433A (zh) 2011-06-23 2011-06-23 逆转或降低食管癌放疗抗性的增敏剂、筛选方法及其用途

Publications (1)

Publication Number Publication Date
WO2012175052A1 true WO2012175052A1 (zh) 2012-12-27

Family

ID=47364331

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/077434 Ceased WO2012175052A1 (zh) 2011-06-23 2012-06-25 逆转或降低食管癌放疗抗性的增敏剂、筛选方法及其用途

Country Status (2)

Country Link
CN (1) CN102836433A (zh)
WO (1) WO2012175052A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112904026A (zh) * 2019-12-03 2021-06-04 深圳艾欣达伟医药科技有限公司 通过前列腺素含量来关联akr1c3酶表达水平及筛选给药用途
CN111214659B (zh) * 2020-02-10 2023-02-10 复旦大学附属肿瘤医院 Cbx4抑制剂在制备食管鳞癌放射增敏剂中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006130666A2 (en) * 2005-06-02 2006-12-07 Schering Corporation Medicaments and methods combining a hcv protease inhibitor and an akr competitor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006130666A2 (en) * 2005-06-02 2006-12-07 Schering Corporation Medicaments and methods combining a hcv protease inhibitor and an akr competitor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DOWNS, T.M.: "PTHrP stimulates prostate cancer cell growth and upregulates aldo-keto reductase 1C3", CANCER LETT., vol. 306, no. 1, 27 March 2011 (2011-03-27), pages 52 - 59 *
XUE, XIAOYING ET AL.: "A study of the relationship of the radioresistance of esophageal carcinoma cell line TE13R120 with HDAC3, NF-kB and NRAGE", CHINESE JOURNAL OF RADIOLOGICAL MEDICINE AND PROTECTION, vol. 25, no. 1, 28 February 2005 (2005-02-28), pages 27 - 30 *

Also Published As

Publication number Publication date
CN102836433A (zh) 2012-12-26

Similar Documents

Publication Publication Date Title
Xun et al. Cancer-derived exosomal miR-138-5p modulates polarization of tumor-associated macrophages through inhibition of KDM6B
Tian et al. Upregulation of long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) is associated with cisplatin resistance in non-small cell lung cancer (NSCLC) by downregulating microRNA-144-3p
Li et al. Overexpression of microRNA-30a-5p inhibits liver cancer cell proliferation and induces apoptosis by targeting MTDH/PTEN/AKT pathway
Subtil et al. Carbon ion radiotherapy of human lung cancer attenuates HIF‐1 signaling and acts with considerably enhanced therapeutic efficiency
Sun et al. Lactate activates CCL18 expression via H3K18 lactylation in macrophages to promote tumorigenesis of ovarian cancer: Role and mechanism of lactate in ovarian cancer
Pei et al. Circular RNA circ-ZEB1 acts as an oncogene in triple negative breast cancer via sponging miR-448
Zhang et al. circRNA_0005529 facilitates growth and metastasis of gastric cancer via regulating miR-527/Sp1 axis
Palmer et al. Molecular markers to predict clinical outcome and radiation induced toxicity in lung cancer
Wang et al. USF1-ATRAP-PBX3 axis promote breast cancer glycolysis and malignant phenotype by activating AKT/mTOR signaling
Zhu et al. Exosomal miR-552-5p promotes tumorigenesis and disease progression via the PTEN/TOB1 axis in gastric cancer
Lin et al. Nucleophosmin/B23 promotes endometrial cancer cell escape from macrophage phagocytosis by increasing CD24 expression
Luo et al. Hsa_Circ_0098181 suppresses hepatocellular carcinoma by sponging miR-18a-3p and targeting PPARA
Niu et al. Effects of USP7 on radiation sensitivity through p53 pathway in laryngeal squamous cell carcinoma
Ren et al. Ultrasound microbubble-stimulated miR-145-5p inhibits malignant behaviors of breast cancer cells by targeting ACTG1
Zhang et al. Exosomal microRNAs in lung cancer: a narrative review
Yang et al. MALAT1 promotes tumorigenesis and increases cellular sensitivity to herceptin in HER2-positive breast cancer
Yan et al. HCG15 is a hypoxia-responsive lncRNA and facilitates hepatocellular carcinoma cell proliferation and invasion by enhancing ZNF641 transcription
Wang et al. shRNA-mediated silencing of Y-box binding protein-1 (YB-1) suppresses growth of neuroblastoma cell SH-SY5Y in vitro and in vivo
Wang et al. Chemosensitizing effect of shRNA-mediated ERCC1 silencing on a Xuanwei lung adenocarcinoma cell line and its clinical significance
Kuang et al. Silencing of circ_002136 sensitizes gastric cancer to paclitaxel by targeting the miR-16-5p/HMGA1 axis
Zhu et al. FOXO4 suppresses cisplatin resistance of triple-negative breast cancer by inhibiting autophagy
Wang et al. Coptisine-mediated downregulation of E2F7 induces G2/M phase arrest in hepatocellular carcinoma cells through inhibition of E2F4/NFYA/NFYB transcription factors
Hou et al. CREB3 facilitates Donafenib resistance in hepatocellular carcinoma cells via the LSD1/CoREST/p65 axis by transcriptionally activating long noncoding RNA ZFAS1
WO2012175052A1 (zh) 逆转或降低食管癌放疗抗性的增敏剂、筛选方法及其用途
CN110251529A (zh) miR-124-3p与其类似物在制备抗乳腺癌疾病药物中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12803330

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 01/04/2014)

122 Ep: pct application non-entry in european phase

Ref document number: 12803330

Country of ref document: EP

Kind code of ref document: A1