CN114344305A

CN114344305A - Application of CDK7 inhibitor THZ1 in radiotherapy resistant treatment of nasopharyngeal carcinoma

Info

Publication number: CN114344305A
Application number: CN202210090425.0A
Authority: CN
Inventors: 袁凯; 刘虹璐; 俞春红; 张娜; 孟阳; 黄璨华; 陈芳
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2021-11-30
Filing date: 2022-01-25
Publication date: 2022-04-15

Abstract

The invention relates to the technical field of biology, in particular to application of a CDK7 inhibitor THZ1 in radiotherapy resistant treatment of nasopharyngeal carcinoma. The invention firstly explores the difference of the phosphorylation levels of the RNA polymerase II C-terminal repetitive domain S5 in nasopharyngeal carcinoma radiotheraphy resistant and radiotheraphy sensitive cells and after the radiotheraphy irradiation. The inhibitor THZ1 of CDK7 target is adopted to detect the drug sensitivity of nasopharyngeal carcinoma radiotherapy resistant and sensitive cells. THZ1 and radiotherapy are combined to act on radiotherapy resistant cells, and whether THZ1 can increase the sensitivity of radiotherapy is researched. THZ1 is applied to a radiotherapy resistant mouse model, and the treatment effect of THZ1 on radiotherapy resistant tumors is explored. The CDK7 inhibitor THZ1 can effectively inhibit the growth of nasopharyngeal carcinoma radiotherapy resistant tumors in-vitro cell experiments and in-vivo nude mouse subcutaneous tumor experiments. From cellular and animal levels, THZ1 was shown to increase sensitivity to radiation therapy when used in combination with radiation therapy, indicating that THZ1 is feasible for the treatment of nasopharyngeal carcinoma against radiation therapy.

Description

Application of CDK7 inhibitor THZ1 in radiotherapy resistant treatment of nasopharyngeal carcinoma

Technical Field

The invention relates to the technical field of biology, in particular to application of a CDK7 inhibitor THZ1 in radiotherapy resistant treatment of nasopharyngeal carcinoma.

Background

Tumors are the main cause of death in China, and the incidence and the death rate of tumors in China keep rising trend in the past half century. In 2018, about 430 ten thousand new cases of tumors and about 290 ten thousand death cases occur. How to effectively treat the tumor is a public health problem to be solved urgently.

The treatment effect of the nasopharyngeal carcinoma is greatly improved by the research of the treatment strategy of the nasopharyngeal carcinoma, but the nasopharyngeal carcinoma patients still have recurrence and metastasis due to radiotherapy resistance in 20-30 percent, and the radiotherapy resistance is considered as a main obstacle of clinical tumor treatment. Current platinum-based dual chemotherapy regimens are used as the first line treatment for recurrent and metastatic nasopharyngeal carcinoma. The research on the mechanism of nasopharyngeal carcinoma radiotherapy resistance mainly relates to the repair and enhancement of DNA damage, cell cycle change, immune escape and the like. It is not clear how the transcriptional changes resulting from radiation therapy cause various lesions in genomic DNA that affect the effectiveness of radiation therapy.

Disclosure of Invention

Technical problem to be solved

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a CDK7 inhibitor, THZ1, for use in the treatment of radiotherapeutic resistance to nasopharyngeal carcinoma, providing a powerful means for the treatment of nasopharyngeal carcinoma.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that: use of the inhibitor THZ1 of CDK7 in the preparation of a medicament for the treatment of nasopharyngeal carcinoma.

Preferably, the inhibitor THZ1 has the chemical formula

Preferably, the inhibitor THZ1 is present at a concentration of 150nM in vitro for a period of 5 days in vitro.

Preferably, mice with the inhibitor THZ1 are administered at a daily dose of 10mg/kg and mice with the inhibitor THZ1 are administered for a period of 26 days.

In another aspect, the invention provides the use of an inhibitor of CDK7, THZ1, to increase the sensitivity of radiotherapy to nasopharyngeal carcinoma.

In addition, the invention also provides a design method of the tumor drug, which takes the subunit CDK7 of TFIIH as a target and develops the inhibitor THZ1 into a drug for treating the tumor resistant to radiotherapy.

Preferably, the tumor is nasopharyngeal carcinoma.

(III) advantageous effects

The invention has the beneficial effects that: the CDK7 inhibitor THZ1 can effectively inhibit the growth of nasopharyngeal carcinoma radiotherapy resistant tumors in-vitro cell experiments and in-vivo nude mouse subcutaneous tumor experiments. From cellular and animal levels, THZ1 was shown to increase sensitivity to radiation therapy when used in combination with radiation therapy, indicating that THZ1 is feasible for the treatment of nasopharyngeal carcinoma against radiation therapy.

Drawings

FIG. 1 is a schematic representation of RNA polymerase II CTD phosphorylation during transcription.

FIG. 2A shows Western blot to detect the levels of S5 and S2 phosphorylation of RNA polymerase II in CNE2 and CNE2-IR cells, with α -tubulin as a control.

FIG. 2B shows Western blot to examine the level of S5 phosphorylation of RNA polymerase II in CNE2 and CNE2-IR cells after exposure to the indicated doses.

FIG. 2C is a quantification of the level of S5 phosphorylation of RNA polymerase II by cells following irradiation. Differences were compared by t-test, indicating p < 0.01.

FIG. 3 shows that nasopharyngeal carcinoma CNE2-IR radiation resistant cells are more sensitive to the CDK7 inhibitor THZ 1.

FIG. 4 shows that THZ1 is effective in reducing the resistance of CNE2-IR cells to radiation therapy.

FIG. 5 shows that THZ1 can reduce the increase in S5 phosphorylation of RNA polymerase II in cells after irradiation with radiation therapy and in cells resistant to radiation therapy.

Fig. 6 is a schematic diagram of an experimental procedure for detecting resistance to radiation therapy in a xenograft tumor model.

FIG. 7 shows the change in tumor volume after radiotherapy in different experimental groups. Differences were compared by t-test, indicating p <0.05 and p < 0.01.

FIG. 8 is an immunofluorescence staining chart of frozen sections of mouse subcutaneous tumors.

FIG. 8A shows that THZ1 is effective in reducing the increase in phosphorylation of RNA polymerase II S5 in mice caused by radiation therapy. RNAPII pS5 is green and DAPI is blue. The scale bar is 1000 μm, and the lower right corner is 10 μm.

FIG. 8B is a comparison of the fluorescence intensity of RNA polymerase II S5 phosphorylation. Differences were compared by t-test, which indicates p < 0.001.

Detailed Description

For a better understanding of the present invention, reference will now be made in detail to the present invention by way of specific embodiments thereof.

The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Unless otherwise indicated, the test methods, detection methods, and preparation methods disclosed herein are those conventionally employed in the relevant fields of molecular biology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, and the like.

Materials, reagents, and the like used in the following examples, unless otherwise described: in particular, they are commercially available.

Gene transcription is mainly divided into four stages: transcription initiation, promoter proximal pause, transcription extension, and transcription termination. When RNA polymerase II is suspended proximal to the promoter, Ser5 of the C-terminal domain (CTD) is phosphorylated by the subunit kinase CDK7 of TFIIH, whereas when RNA polymerase II is released upon suspension into transcriptional elongation, Ser2 is phosphorylated by the subunit CDK9 of P-TEFb, as shown in fig. 1.

Tumor carcinogenic transcription factors can be indirectly inhibited by targeting enzyme accessory factors such as cyclin-dependent kinase and the like. THZ1, an anilinopyrimidine, inhibits the activity of CDK7 by covalent modification, as shown in the following chemical formula. THZ1 is effective against the proliferative activity of T-ALL cells and other hematologic malignancies. In Jurkat cells, low doses of THZ1 can affect a small portion of genes such as the key regulatory factor RUNX1, leading to gene expression deregulation and cell death.

Experimental object of embodiment of the invention

Nasopharyngeal carcinoma cells were collected from the sources shown in the table below; 24 BALB/c-nu nude mice of 4 weeks old were purchased from Jiangsu Jiejiaokang Biotechnology GmbH.

Cell name	Tissue or disease class	Source
			CNE2	Nasopharyngeal carcinoma	Shishishiqiang teacher in Hunan ya Hospital of China-south university present
CNE2-IR	Nasopharyngeal carcinoma	Shishishiqiang teacher in Hunan ya Hospital of China-south university present

Example 1

The phosphorylation level of RNAPII CTD is different in sensitive and resistant cells of nasopharyngeal carcinoma radiotherapy.

According to the embodiment, the following steps are carried out: difference in phosphorylation of RNA polymerase II Ser5 in nasopharyngeal carcinoma radiotherapy-sensitive cells and radiotherapy-resistant cells.

(1) Materials and reagents:

basal media and serum as shown in the following table were purchased and prepared in a proportion of 10% FBS for culturing cells such as CNE2 and CNE 2-IR. The protein was collected from the cell lysate and subjected to western blotting.

(2) Experimental procedure

1) Collecting the protein

Will be 4X 10⁵The individual cells were plated in 6-well plates at 37 ℃ and 5% CO₂Incubate for 24 hours in ambient. The medium was removed, the cells were rinsed 3 times with buffer DPBS, 200. mu.l of cell lysate (2% SDS, 10% glycerol, 62.5mM Tris-HCl pH 6.8; 1 Xprotease inhibitor cocktail,10mM sodium fluoride, 1mM sodium orthovanadate) was added, the cells were scraped off with a cell spatula, treated on a dry heat instrument at 95-100 ℃ for 5 minutes, and the pellet was removed after spotting.

2) Ionizing radiation

Will be 4X 10⁵The cells were plated in 6-well plates and cultured at 37 ℃ in a 5% CO2 atmosphere for 24 hours, followed by linear accelerator (X-Rad 225)Precision), irradiation was performed at room temperature and 200cGy/min at different doses, and the protein was collected after further culturing for 48 hours, as described above.

3) Determination of protein concentration

1 piece of 96-well plate is taken, and the protein standard is diluted according to the concentration gradient of 2,1.5,1,0.75,0.5,0.25,0.125 and 0.05mg/ml, and 6 mu l of protein standard is added into each well. Add 4 microliters of the cellular protein sample and 2 microliters of sterile deionized water. The standard and the sample are both provided with two complex holes. BCA reagent A and BCA reagent B are prepared into BCA working solution according to the ratio of 50:1, 120 mu l of BCA working solution is added into each well, and the mixture is incubated for 20-30 minutes at 37 ℃ in a dark place. A562 was measured with a microplate reader, and the protein concentration of the sample was calculated from the standard curve and the sample volume used.

4) Western blot experiment

Step 1: cleaning glass plate, preparing 8% separation gel, mixing, rapidly adding into glass plate, slowly adding ddH₂And O, flattening the glue interface.

Reagents or materials	Volume of
		ddH₂O	4.6ml
30% acrylamide	2.6ml
		1.5M Tris-HCl pH 8.8	2.6ml
10％SDS	100μl
		10％APS	100μl
TEMED	10μl

Step 2: after the separation gel is solidified, 4% of concentrated gel is prepared and a comb which is suitable for the specification of the glass plate is inserted.

And 3, step 3: after the gel is solidified, the comb is slowly and vertically pulled out. Placing the glass plate into a gel running electrophoresis box, preparing 10X electrophoresis liquid, and adding 1X electrophoresis liquid into the electrophoresis box.

Reagents or materials	Volume or weight
		glysin	144g
Tris	30g
		SDS	10g
ddH₂O	to 1L

And 4, step 4: the sample loading volume was calculated as 20 μ g per well, and the volume was calculated as 10: 1 adding 10 × loading buffer (1% bromophenol blue + β -mercaptoethanol ═ 1:4) to the sample, treating at 95-100 ℃ for 5 minutes on a dry heat instrument, spotting, and mixing.

And 5, step 5: and (4) loading. Samples were spotted one by one on a protein Marker and sample basis, and the remaining wells were filled with 1 × loading buffer.

And 6, step 6: and (6) glue running. The sample was run at 80V for 30 minutes and continued at 120V for 1 hour after the sample had been aligned to one line.

And 7, step 7: and (5) transferring the film. Preparing 1 x membrane transferring liquid, and clamping a membrane transferring bottom plate, a sponge, filter paper, separation gel, a PVDF membrane, filter paper, a sponge and a transparent top plate in sequence in a container filled with the membrane transferring liquid. Under the low temperature environment, the temperature is changed for 90 minutes by a constant current of 290 mA.

Reagents or materials	Volume or weight
		glysin	9g
Tris	1.93g
		Methanol	200ml
ddH₂O	to 1L

And 8, step 8: and (5) sealing. The PVDF membrane is taken out, washed by PBST, and then put into PBST (0.1 percent TritonX-100) to prepare 5 percent skim milk, and the phosphorylation of the protein needs to be detected by blocking with 5 percent BSA. The incubation was performed on a shaker with the membrane facing up and at room temperature for 1 hour.

Step 9: primary antibody incubation. The milk-blocked membranes were washed 3 times with PBST and incubated overnight at 4 ℃ on a shaker with RNAPII antibody (murine antibody, 1:3000), RNAPII S5 phosphorylated antibody (murine antibody, 1:3000), and RNAPII S2 phosphorylated antibody (rabbit antibody, 1:5000), alpha-Tubulin (murine antibody, 1:5000) formulated with 5% BSA.

Step 10: and (5) incubating a secondary antibody. PBST was washed 3 times, and incubated with a secondary antibody formulated with 5% BSA (1: 5000) on a shaker at room temperature for 1 hour.

And 11, step 11: and (6) developing. After PBST 3 washes, ECL was developed.

(3) Test results and conclusions:

in nasopharyngeal carcinoma radiation sensitive and resistant cells, RNAPII CTD levels of Ser5 phosphorylation were higher in resistant cells and Ser2 phosphorylation were lower in resistant cells relative to the protein level of total RNAPII, as shown in figure 2A. After a dose of irradiation, both CNE2 and CNE2-IR showed increased levels of RNAPII Ser5 phosphorylation, and CNE2-IR showed higher levels of RNAPII Ser5 phosphorylation than CNE2, as shown in fig. 2B, and fig. 2C is a grayscale histogram of three replicates.

Example 2

CNE2 and CNE2-IR differ in their sensitivity to the CDK7 inhibitor THZ 1.

(1) Materials and reagents:

the following reagents were purchased.

(2) Experimental procedure

1) Drug treatment

Step 1: taking a 10cm culture dish as an example, digesting logarithmic phase cells with 1ml of pancreatin, centrifuging and collecting after terminating digestion, re-suspending into cell suspension by using phenol red-free DMEM medium, counting, and then, according to 1000 CNE2 cells per well and 1200 CNE2-IR cells per well, after counting, paving an appropriate amount of cells in a 96-well plate (the marginal wells are filled with sterile PBS), wherein more than three wells are arranged at each dosing concentration. Three replicate wells were kept with only an equal amount of medium as background and cultured at 37 ℃ and 5% CO2 until the cell monolayer was confluent at the bottom of the well.

Step 2: the drugs are dissolved in 10% DMSO DPBS buffer solution, after the cells adhere to the wall, the drugs are added according to concentration gradients of 0, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 mu M, 0 mu M of control is set as that only equal volume of solvent is added, and the DMSO final concentration of each well is less than 0.2%. The cells were cultured for an additional 3 days at 37 ℃ and 5% CO 2.

2) MTT (methyl thiazolyl tetrazolium) experiment detection cell viability

Step 1: mu.l of MTT solution (5mg/ml, i.e., 0.5% MTT) was added to each well, and the culture was terminated after 4 hours of further culture.

Step 2: mu.l of triple lysis solution (10% SDS, 5% isobutanol, 0.012mol/l concentrated HCl) was added to each well and incubated overnight in an incubator.

And 3, step 3: and (3) detecting by using a microplate reader, treating the cells with the drug for 3 days, and recording the absorbance values of the cells of the control group (solvent) and the cells of the experimental group (drug) at 490nm wavelength.

And 4, step 4: subtracting the background value from the absorbance value of each well, averaging the absorbance value of each concentration of the experimental group, dividing by the average absorbance value of the control group to obtain the average cell survival rate (%) under the action of different drug concentrations, and calculating the standard deviation under different drug concentrations. Cell growth curves were plotted with the log of drug concentration as the abscissa and the mean cell survival (%) as the ordinate, see FIG. 3, and the IC50 was calculated using GraphPad for Dose-response-Inhibition parameter analysis.

(3) Test results and conclusions:

CNE2-IR radiation therapy resistant cells were more sensitive to the CDK7 inhibitor THZ1 with an IC50 of 209 nM. The IC50 of CNE2 cells was 1027 nM.

Example 3

Effect of radiotherapy irradiation and THZ1 on growth proliferation of CNE2 and CNE 2-IR.

(1) Reagents and materials:

reference is made to example 2.

(2) The experimental steps are as follows:

1) drug treatment

Taking CNE2 as an example, 4X 105 cells per well were plated in 2 wells of a 6-well plate, plated in 2 blocks, and cultured at 37 ℃ in a 5% CO2 environment to allow the cells to adhere to the wall. The drugs were dissolved in 10% DMSO DPBS buffer, 150nM THZ1 was added to each plate, and 0. mu.M THZ 1-free solvent was added to each plate, with a final DMSO concentration of less than 0.2% per well, and incubated at 37 ℃ and 5% CO2 for 12 hours.

2) Ionizing radiation

1 of these 6-well plates was irradiated to 2Gy dose at room temperature and 200cGy/min using a linear accelerator (X-Rad 225, Precision). CNE2 cells of a control group (solvent, 0Gy) and an experimental group (drug, 0 Gy; solvent, 2 Gy; drug, 2Gy) are digested, centrifuged and resuspended in phenol red-free medium, after counting, the CNE2 cells are paved on 96-well plates according to 1000 per well, each group has at least 3 duplicate wells, three duplicate wells are reserved, only the same amount of medium is added as background, and the CNE2 cells are cultured for 5 days at 37 ℃ and in a 5% CO2 environment. CNE2-IR was treated as in CNE2, 1200 per well in 96 well plates.

3) MTT (methyl thiazolyl tetrazolium) experiment detection cell viability

Refer to Steps 1-2 of experiment 2. After the cells were examined by a microplate reader and treated with radiotherapy and the drug together for 5 days, the absorbance values of the cells in the control group (solvent, 0Gy) and the experimental group (drug, 0 Gy; solvent, 2 Gy; drug, 2Gy) at 490nm wavelength in each well were recorded. Subtracting the background value from the absorbance value of each well, calculating the ratio of the absorbance values of the experimental group and the control group respectively to obtain the cell survival rate (%) of the experimental group under different conditions, and drawing the histogram of fig. 4.

(3) Results and conclusions:

as shown in FIG. 4, CNE2-IR cells treated with the inhibitor THZ1 after 2Gy of radiotherapy can effectively reduce the resistance of CNE2-IR cells to radiotherapy and have the effect of increasing the sensitivity of radiotherapy compared with CNE2-IR cells without THZ 1.

Example 4

Changes in RNAPII Ser5 phosphorylation in CNE2 and CNE2-IR cells under the combined action of radiation therapy irradiation and THZ 1.

(1) Materials and reagents:

reference is made to experiment 1 and experiment 2.

(2) The experimental steps are as follows:

1) adding chemicals for treatment

Taking CNE2 as an example, 4X 105 cells per well were plated in 2 wells of a 6-well plate, plated in 2 blocks, and cultured at 37 ℃ in a 5% CO2 environment to allow the cells to adhere to the wall. Drugs were dissolved in 10% DMSO DPBS buffer, and THZ1 was added to each plate at a final concentration of 250nM, and 0. mu.M THZ 1-free solvent, at a final DMSO concentration of less than 0.2% per well, and incubated at 37 ℃ and 5% CO2 for 12 hours.

2) Ionizing radiation

1 of the 6-well plates was irradiated with a linear accelerator (X-Rad 225, Precision) at room temperature and 200cGy/min to a dose of 6Gy, and incubated at 37 ℃ and 5% CO2 for 48 hours. CNE2-IR was treated as in CNE 2.

Collecting the protein

The control group (solvent, 0Gy) and the experimental group (solvent, 6 Gy; drug, 0 Gy; drug, 6Gy) were subjected to the procedure of experiment 1 to collect proteins.

4) Western blot experiment

Refer to the experimental procedure of experiment 1.

Results and conclusions:

as shown in fig. 5, THZ1 can reduce the level of RNAPII Ser5 phosphorylation of CNE2-IR compared to CNE 2. And THZ1 can reduce increased phosphorylation of RNAPII Ser5 in CNE2 and CNE2-IR due to radiation exposure.

Example 5

The THZ1 inhibitor can be added to effectively inhibit the growth of nasopharyngeal carcinoma radiotherapy resistant cell xenograft tumor.

(1) Materials and reagents:

24 BALB/c-nu nude mice, 4 weeks old, were purchased from Schlekschada laboratory animals Co., Ltd, Hunan, and two weeks later, experiments were conducted.

(2) The experimental steps are as follows:

step 1: CNE2-IR cells were harvested by digestion, and after cell counting, the cells were resuspended in DPBS and adjusted to a cell concentration of 5X 107/ml. Then, 5X 106 cells in 100. mu.l were inoculated subcutaneously into nude mice by aspiration with an insulin needle.

Step 2: grouping was performed when the mean tumor volume reached 200mm 3. As shown in fig. 6, the method is divided into: control group, solute injection and 4Gy radiotherapy group, THZ1 injection and 4Gy radiotherapy group.

And 3, step 3: a10% DMSO solution prepared from 5% medical glucose injection was used as a solute, and THZ1 was prepared as a mother solution at 2 mg/ml. The THZ1 injection groups were each administered with 10mg/kg/day intraperitoneal THZ1 using a 1ml syringe, the solute injection group was administered with 10mg/kg/day of solute under the same conditions, and the drug injection was maintained until the termination of the experiment.

And 4, step 4: preparing the anesthetic. Adding 10mg tribromoethanol to 10ml tert-amyl alcohol for complete dissolution to prepare mother liquor, and packaging with tinfoil paper for storage at 4 deg.C in dark place. According to the following steps: 40 dissolving the mother solution in normal saline, filtering with 0.22 μm filter membrane, storing at 4 deg.C in dark place, and keeping the anesthetic effective period of 1 month.

And 5, step 5: the mice in the THZ1 group and solute group were subjected to intraperitoneal injection at an anesthetic injection amount of 10 to 20. mu.l/g 24 hours after the initial injection of THZ1 and solute, were anesthetized 5 to 10 minutes later, the bodies of the mice were covered with lead blocks, only the tumor portions were exposed, and were irradiated at a dose of 4Gy at room temperature and 200 cGy/min. After the mice recovered, tumor volumes were measured every other day and growth curves were plotted, as shown in fig. 6.

(3) Results and conclusions:

as shown in FIG. 7, by periodically measuring changes in tumor volume, it was found that tumor proliferation was restored after the growth of the CNE2-IR resistant tumor was inhibited for a short period of days after 4Gy radiotherapy treatment, as compared with the control group. And the THZ1 medicine can effectively inhibit the proliferation of the resistant tumor after 4Gy radiotherapy treatment. It is shown that THZ1 is also effective in inhibiting the growth of tumors resistant to radiotherapy in mice.

Example 6

THZ1 is effective in inhibiting increase of phosphorylation level of RNAPII Ser5 caused by radiotherapy in vivo. (1) Materials and reagents: the following reagents were purchased.

(2) The experimental steps are as follows:

1) tumor tissue exfoliation and dehydration

Step 1: mice were sacrificed by cervical dislocation, tumors were dissected off on a clean bench using sterile blades, and mouse carcasses were treated as prescribed in the animal room.

Step 2: the tumor was quickly placed in a diluted 4% formaldehyde solution and fixed overnight in a refrigerator at 4 ℃.

And 3, step 3: the tumor was washed three times with PBS on a shaker for 10 minutes each.

And 4, step 4: the mass was added to a 15% sucrose solution and placed in a refrigerator at 4 ℃ for 2-3 hours, after which the mass settled to the bottom.

And 5, step 5: the tumor was added to a 30% sucrose solution and refrigerated overnight at 4 ℃.

2) OCT embedding

Step 1: approximately 5cm by 5cm of square tinfoil paper is prepared and folded to form a cuboidal container. And information of the tumor is marked on the side with a Marker pen.

Step 2: adding a layer of OCT reagent at the bottom, adding the tumor into a marked tinfoil paper container, and quickly adding a layer of OCT on the tumor until the tumor tissue is completely covered. And the tissue was quickly snap frozen in liquid nitrogen using forceps.

And 3, step 3: and after OCT is completely whitened, taking out and wrapping the tin foil paper, and immediately storing at-80 ℃.

3) Frozen sections and immunofluorescence staining

Step 1: precooling a German Leica 1950 type quick freezing slicer to-20 ℃, taking out a tissue extractor, and fixing the tissue on the extractor by using OCT.

Step 2: the supporting device is fixed on the holding device, the advancing and retreating key is started, the knob is rotated, and the tissue is flattened.

And 3, step 3: the thickness to be cut is adjusted to about 12um, the anti-rolling plate is adjusted, the cut section can smoothly pass through the space between the knife and the anti-rolling plate at the first time and is flatly laid on the baffle of the knife holder, at the moment, the anti-rolling plate can be lifted, a precooled glass slide is taken out and is pasted and marked. The slices were stored in a-80 ℃ freezer.

And 4, step 4: the plates were fixed with acetone for 5 minutes at room temperature and washed 2 times with PBS for 5 minutes each.

And 5, step 5: blocking with 5% BSA at room temperature for 30 min.

And 6, step 6: BSA was aspirated and incubated with RNAPII S5 phosphorylated antibody (murine antibody, 1:1000) overnight at 4 ℃ in the refrigerator.

And 7, step 7: PBST was washed 3 times for 10 minutes each, and a secondary antibody Alexa Fluor 488dye (1: 500) was incubated for 1 hour at room temperature in the dark.

And 8, step 8: PBST was washed 3 times and stained with DAPI for 1 min. PBST was washed 3 times and mounted.

Step 9: microscopic imaging was performed using ZESIS LSM 880 confocal.

(3) Results and conclusions: the mice of a control group, a solute injection and 4Gy radiotherapy group, a THZ1 injection and 4Gy radiotherapy group are subjected to immunofluorescence staining of RNAPII Ser5 phosphorylation on tumor tissues of the mice in a frozen section, and the result shows that compared with the control group, the phosphorylation of RNAPII Ser5 of the tumor treated by 4Gy radiotherapy is increased, and the phosphorylation increase of RNAPII Ser5 of the tumor after 4Gy radiotherapy treatment can be effectively reduced after the THZ1 medicine is added. Indicating that THZ1 was also effective in reducing the increase in RNAPII Ser5 phosphorylation caused by radiation therapy in mice, see figure 8.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of THZ1 inhibitor CDK7 in preparing medicine for treating nasopharyngeal carcinoma is disclosed.

2. Use of the inhibitor THZ1 of CDK7 in the preparation of a medicament for the treatment of nasopharyngeal carcinoma according to claim 1, wherein the inhibitor THZ1 has the formula

3. Use of the inhibitor THZ1 of CDK7 according to claim 1 or claim 2 in the preparation of a medicament for the treatment of nasopharyngeal carcinoma, wherein the inhibitor THZ1 is present at a concentration of 150nM in vitro for a period of 5 days in vitro.

4. Use of the inhibitor THZ1 of CDK7 according to claim 1 or claim 2 in the preparation of a medicament for the treatment of nasopharyngeal carcinoma, wherein the mouse of the inhibitor THZ1 is administered at a daily dose of 10mg/kg and the mouse of the inhibitor THZ1 is administered for a period of 26 days.

5. Use of an inhibitor of CDK7, THZ1, for increasing the sensitivity of nasopharyngeal carcinoma to radiation therapy.

6. A method for designing a tumor drug is characterized in that a subunit CDK7 of TFIIH is taken as a target, and an inhibitor THZ1 is used for developing a drug for treating tumors resistant to radiotherapy.

7. The method of claim 6, wherein the tumor is nasopharyngeal carcinoma.