CN116064799B

CN116064799B - Application of AKR1C1 in the preparation of diagnostic and therapeutic products for extrahepatic bile duct cancer

Info

Publication number: CN116064799B
Application number: CN202211100871.1A
Authority: CN
Inventors: 周琳; 刘畅; 吴洪坤; 任鹤菲; 陈赛; 张小敏; 张嘉丰
Original assignee: Shanghai Changzheng Hospital
Current assignee: Shanghai Changzheng Hospital
Priority date: 2022-09-09
Filing date: 2022-09-09
Publication date: 2025-10-28
Anticipated expiration: 2042-09-09
Also published as: CN116064799A

Abstract

The invention relates to the technical field of medical biological detection, and provides a new application of AKR1C1, in particular to an application in preparing reagents or kits for diagnosis, stage classification and prognosis evaluation of extrahepatic bile duct cancer, and the diagnosis, stage classification and prognosis evaluation of extrahepatic bile duct cancer are realized by detecting the expression quantity of AKR1C1 in a biological sample; the invention further provides application of the agent for inhibiting or silencing AKR1C1 expression in preparing extrahepatic cholangiocarcinoma medicines, which can inhibit proliferation and migration capacity of extrahepatic cholangiocarcinoma cells and promote death of tumor cells by targeting down-regulating AKR1C1 expression, and finally improve or treat extrahepatic cholangiocarcinoma.

Description

Application of AKR1C1 in preparation of extrahepatic cholangiocarcinoma diagnosis and treatment product

Technical Field

The invention relates to the technical field of biological detection, relates to application of AKR1C1 as an extrahepatic bile duct cancer biomarker, and in particular relates to application of AKR1C1 in preparation of extrahepatic bile duct cancer diagnostic reagents or kits and therapeutic pharmaceutical compositions.

Background

Extrahepatic cholangiocarcinoma (extrahepatic cholangiocarcinoma, ECC) refers to a malignancy originating from extrahepatic cholangiocellular epithelial cells, with an incidence of about 90% of cholangiocarcinoma. Extrahepatic bile duct cancer is frequently misdiagnosed and mishandled due to complex and diverse clinical courses and lack of typical symptoms, and simultaneously, the prognosis of the cancer is extremely poor due to high recurrence rate and metastasis rate, the survival rate in 5 years is about 5%, and the disease seriously endangers the health of human beings. Moreover, the pathogenesis of the etiology is unknown so far, and no specific diagnosis means and no ideal treatment method exist at home and abroad. Therefore, the method is of great significance in searching the occurrence mechanism of extrahepatic bile duct cancer, searching accurate and effective early diagnosis and treatment targets, delaying disease progression, improving survival rate of patients and improving prognosis.

The aldehyde ketoreductase family (Aldo-keto reductases, AKRs) is a superfamily containing 16 families, and it is found that more than 190 family members are involved in redox reactions with nicotinamide adenine dinucleotide phosphate (triphosphopyridine nucleotide, NADPH) as a coenzyme, and is one of three enzyme superfamilies of redox reactions, widely distributed in prokaryotes and eukaryotes such as plants, fungi and vertebrates. These 16 families are called AKR1-AKR16, respectively, but only three families, AKR1, AKR6, AKR7, belong to the mammalian family, AKR1 being the largest family. Related studies have shown that AKR1C plays an important role in the development and Drug resistance of tumors (PENNING TM et al, aldo-Keto Reductases AND CANCER Drug resistance. Pharmacol Rev.2021,73 (3): 1150-1171.Zeng CM et al ,Aldo-Keto Reductase AKR1C1-AKR1C4:Functions,Regulation,and Intervention for Anti-cancer Therapy.Front Pharmacol.2017,14;8:119.).

AKR1C comprises AKR1C1-C4 subtypes, wherein AKR1C4 is mainly liver-specific. Although AKR1C is a reductase, more and more studies have found that AKR1C1-C3 has a non-catalytic effect in tumor cells, including the function as a coactivator (Park S et al ,Inhibitory Interplay of SULT2B1b Sulfotransferase with AKR1C3 Aldo-keto Reductase in Prostate Cancer.Endocrinology.2020,1;161(2):bqz042.)、 regulates E3-ligase-ubiquitin system (Fan L,The steroidogenic enzyme AKR1C3 regulates stability of the ubiquitin ligase Siah2 in prostate cancer cells.J.Biol.Chem.2015,21;290(34):20865–20879.)、 to regulate tumor drug sensitivity (Phoo NLL et al ,Transcriptomic Profiling Reveals AKR1C1 and AKR1C3 Mediate Cisplatin Resistance in Signet Ring Cell Gastric Carcinoma via Autophagic Cell Death.Int J Mol Sci.2021,19;22(22):12512.)、 tumor cells apoptosis (Li C et al ,Panax ginseng polysaccharide induces apoptosis by targeting Twist/AKR1C2/NF-1pathway in human gastric cancer.Carbohydr Polym.2014,15;102:103-9.) and metastasis (Zhu H et al ,AKR1C1 Activates STAT3 to Promote the Metastasis of Non-Small Cell Lung Cancer.Theranostics.2018,1;8(3):676-692.). Et al.) however, no relevant report has been made regarding the effect of AKR1C1-C4 in extrahepatic cholangiocarcinoma. Then, whether there is an abnormality in the expression level of AKR1C1-C4 in ECC, whether the differentially expressed AKR1C molecule is associated with the progress of ECC, whether it can regulate the effect of ECC cells on proliferation, death, and migration ability, etc. is still unclear.

Disclosure of Invention

The invention is carried out by relying on the research, and aims to provide a biomarker for diagnosis of extrahepatic bile duct cancer and also provides a new application of AKR1C1, namely application in preparation of an extrahepatic bile duct cancer diagnosis kit or a therapeutic pharmaceutical composition.

The invention firstly starts from intrahepatic and extrahepatic bile duct cancer cell strains and tumor tissues, and analyzes the expression conditions of AKR1C1-C4, and the results show that AKR1C1 and AKR1C2 are highly expressed in extrahepatic bile duct cancer, then the expression of AKR1C1 and AKR1C2 is detected in digestive system tumor cells, and the results show that AKR1C1 is specifically highly expressed in ECC.

Next, immunohistochemical detection of AKR1C1 expression in tumor tissues and paracancerous control tissues of ECC patients and analysis of its relationship with ECC disease progression and prognosis judgment, and inhibition of differential gene expression in ECC cells, detection of proliferation, death and migration ability of ECC cells. The AKR1C1 is analyzed to be valuable in the aspects of accurate diagnosis and treatment of extrahepatic cholangiocarcinoma, thereby providing effective molecules for diagnosis, treatment and prognosis evaluation for ECC.

Specifically, the invention provides the following technical scheme:

In a first aspect of the invention, there is provided the use of AKR1C1 as a diagnostic marker. In particular to application of a reagent for detecting AKR1C1 in preparing a kit for diagnosis, stage classification and prognosis evaluation of extrahepatic bile duct cancer.

Preferably, the reagent for detecting AKR1C1 is a reagent for detecting the AKR1C1 expression level in a biological sample at a gene level and/or a protein level, and the reagent kit comprises the reagent for detecting the AKR1C1 expression level in the biological sample.

Further preferably, the reagent for detecting the AKR1C1 expression level in the biological sample is selected from one or more of the group consisting of immunohistochemistry, western blot blotting, and qRT-PCR. The three methods can be adopted to realize the detection of AKR1C1 expression in biological samples.

Further preferably, the reagent for detecting the expression level of AKR1C1 in a biological sample comprises a PCR primer having detection specificity for AKR1C1 gene or an antibody specifically binding to AKR1C1 protein. Wherein, the PCR primer with detection specificity to AKR1C1 gene is shown as SEQ ID NO. 1-2, and the primer sequence of the contrast GAPDH is shown as SEQ ID NO.9 and SEQ ID NO. 10.

In a second aspect of the invention, there is provided a kit for extrahepatic cholangiocarcinoma diagnosis, staging or prognosis evaluation, the kit comprising reagents for detecting the AKR1C1 content in a biological sample.

The kit for detecting the protein level consists of a reagent system for preparing paraffin sections of tumor tissues, an antigen repair reagent system and an antibody system, wherein the antibody system is preferably a monoclonal antibody of an AKR1C1 protein.

AKR1C1-F primer AGAATGGCTTTGCTGTGGTC (SEQ ID NO. 1);

AKR1C1-R primer ATCTTGCTCACGCTCAACCT (SEQ ID NO. 2);

GAPDH-F primer CAGGAGGCATTGCTGATGAT (SEQ ID NO. 9);

GAPDH-R primer GAAGGCTGGGGCTCATTT (SEQ ID NO. 10).

Further, the biological sample is selected from any one of surgical tumor tissue, tumor tissue obtained by puncturing or circulating tumor cells in blood of a patient. For early diagnosis and tumor patients losing operation time, the detection of AKR1C1 expression level can be carried out by obtaining cancer tissues through puncture, or the detection of AKR1C1 level can be carried out by collecting circulating tumor cells in blood of the patients, so that early diagnosis and prognosis evaluation can be realized.

In a third aspect of the invention, there is provided the use of a substance which inhibits or silences AKR1C1, i.e. in the manufacture of a medicament for the treatment of extrahepatic cholangiocarcinoma.

Preferably, the substance for inhibiting or silencing AKR1C1 is shRNA for inhibiting AKR1C1 expression or a recombinant expression vector or transgenic cell line containing the shRNA, and the sequence of the shRNA is as follows:

CACCAAATTGGCAATTGAA(SEQ ID NO.11)。

In a fourth aspect, the invention provides a pharmaceutical composition for treating extrahepatic cholangiocarcinoma, which comprises an active component and a pharmaceutically acceptable carrier, wherein the active component comprises shRNA for inhibiting AKR1C1 expression or a recombinant expression vector or a transgenic cell line containing the shRNA, and the sequence of the shRNA is shown as SEQ ID NO. 11.

In a fifth aspect the present invention provides a product comprising at least one of the second, third and fourth aspects. The product has at least one of the functions (1) - (4):

(1) Diagnosing and/or preventing extrahepatic cholangiocarcinoma;

(2) Inhibiting proliferation of extrahepatic bile duct cancer cells;

(3) Promoting extrahepatic bile duct cancer cell death;

(4) Inhibit the migration of extrahepatic bile duct cancer cells.

The extrahepatic bile duct cancer cells are QBC939 cells.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses application of AKR1C1 in extrahepatic bile duct cancer diagnosis or prognosis evaluation for the first time, and the AKR1C1 can be used as a marker for extrahepatic bile duct cancer diagnosis, staging and prognosis evaluation according to the relationship between AKR1C1 and staging, total survival time, no disease progression and lymph node metastasis.

The invention discloses an application of inhibiting AKR1C1 in preventing and treating extrahepatic bile duct cancer for the first time, and can inhibit proliferation and migration capacity of extrahepatic bile duct cancer cells, promote death of tumor cells and finally improve or treat extrahepatic bile duct cancer by targeting and downregulating expression of AKR1C 1.

The invention also provides shRNA targeting AKR1C1 and a recombinant expression vector or a transgenic cell line containing the shRNA, which can inhibit proliferation and migration capacity of extrahepatic bile duct cancer cells and promote death of tumor cells by targeting down-regulating AKR1C1 expression, and finally improve or treat extrahepatic bile duct cancer.

For the detection technology, the detection of AKR1C1 is essentially a quantitative PCR detection based on the expression status of blood cell genes, has the characteristics of simple and convenient operation, sensitive detection, good specificity, high repeatability and the like, and is increasingly applied to clinical detection technology nowadays. The basic detection method adopted by the invention is real-time fluorescence quantitative PCR, the sensitivity and the accuracy of the technology are high, the clinical application is wide, and the test technology is mature.

In terms of effect, the index AKR1C1 related to the invention is specifically and highly expressed in tumor tissues of patients with extrahepatic bile duct cancer, and the difference has statistical significance (P is less than 0.05), can be used as a diagnosis, metastasis and/or prognosis marker of extrahepatic bile duct cancer, and can inhibit proliferation and migration of extrahepatic bile duct cancer cells and promote death of extrahepatic bile duct cancer cells by downregulating AKR1C 1. So that the clinical reference value and the credibility are higher.

Drawings

FIG. 1 shows that AKR1C1 is specifically and highly expressed in extrahepatic cholangiocarcinoma, wherein A is the expression of AKR1C1-C4 in intrahepatic and extrahepatic cholangiocarcinoma cells, B is the expression of AKR1C1-C4 in intrahepatic and extrahepatic cholangiocarcinoma tissues, C is the expression of AKR1C1 and AKR1C2 in digestive system tumor cells, and D is the expression of AKR1C1 in extrahepatic cholangiocarcinoma tissues and paracancerous control normal tissues thereof.

FIG. 2 shows AKR1C1 as a diagnosis, metastasis and/or prognosis marker of extrahepatic cholangiocarcinoma, wherein A is the expression of AKR1C1 of different differentiation degrees of tumor tissue of 55 extrahepatic cholangiocarcinoma patients, B is the total survival of 55 extrahepatic cholangiocarcinoma patients, and FIG. 2C is the non-recurrent survival of 55 extrahepatic cholangiocarcinoma patients.

FIG. 3 shows that down-regulating AKR1C1 can inhibit proliferation and migration of extrahepatic bile duct cancer cells and promote death of extrahepatic bile duct cancer cells, wherein A is efficiency verification of down-regulating AKR1C1 by adding DOX after stably transfecting shRNAAKR C1 of Doxcycline (DOX) regulatory system in extrahepatic bile duct cancer cell strain QBC939, B, C is proliferation condition of QBC939 cells after down-regulating AKR1C1, D, E is death condition of QBC939 cells after down-regulating AKR1C1, and F is migration condition of QBC939 cells after down-regulating AKR1C 1.

Detailed Description

The present invention will now be described in detail with reference to examples and drawings, but the practice of the invention is not limited thereto.

The reagents and starting materials used in the present invention are commercially available or may be prepared by literature procedures. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions as described in Sambrook et al, molecular cloning, laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or under conventional conditions, or under conditions recommended by the manufacturer.

Example 1 AKR1C1 is specifically highly expressed in extrahepatic bile duct cancer

In order to determine the expression and expression specificity of AKR1C1-C4 in intrahepatic cholangiocarcinoma (INTRAHEPATIC CHOLANGIOCARCINOMA, ICC) and extracholangiocarcinoma, the expression of AKR1C1-C4 was detected by a real-time quantitative reverse transcription PCR (qRT-PCR) method in human intrahepatic cholangiocarcinoma cell line HCCC and human extrahepatic cholangiocarcinoma cell line QBC939, and in tumor tissues of intrahepatic and extrahepatic cholangiocarcinoma patients, respectively. HCCC9810, QBC939 and liver cancer cell lines are purchased from Shanghai eastern hepatobiliary surgery Hospital in Shanghai, human esophageal cancer cell line TE-1, pancreatic cancer cell line PANC1, gastric cancer cell line MGC823 and colon cancer cell line HCT8 in Shanghai department of biological science cell banks of China academy of sciences. Tumor specimens of intrahepatic and extrahepatic cholangiocarcinomas patients are both diagnosed by the pathologist as corresponding cancers. The reverse transcription system and conditions are shown in Table 1 below (Takara Co., ltd. Reverse transcription kit):

TABLE 1 reverse transcription System

* The reverse transcription volume can be increased as desired, but the amount of RNA per 10. Mu.l system does not exceed 500ng, increasing proportionally.

The reverse transcription reaction conditions are 37 ℃,15min, 85 ℃,5s, 4 ℃ and infinity.

The RT-PCR system and conditions are shown in Table 2 below (Takara RT-PCR kit):

TABLE 2 RT-PCR System

The amplification conditions are 94 ℃,30s, 55-60 ℃,30s, 72 ℃ and 1min. The three steps were performed for 40 cycles in total.

The primer sequences used in the PCR reaction are shown in Table 3:

TABLE 3 qRT-PCR primer sequences

The results showed that AKR1C1 and AKR1C2 were highly expressed in ECC cells, whereas AKR1C3 and AKR1C4 were expressed indifferently in intrahepatic and extrahepatic cholangiocarcinoma cells, compared to ICC cells (FIG. 1A), and that the results of tumor tissue detection in intrahepatic and extrahepatic cholangiocarcinoma patients were consistent with cells, we also found that AKR1C1 expression was higher in most ECC patients than in ICC patients, but that the expression levels in most ICC and ECC patients were consistent in AKR1C2, and that AKR1C2 expression was significantly higher in only a few ECC cancer patients than in ICC patients (FIG. 1B), suggesting that AKR1C1 was most likely to be specifically highly expressed in ECC.

Then, to further clarify the specificity of AKR1C1, we continued to detect the expression of AKR1C1 and AKR1C2 in tumor cells of the digestive system by qRT-PCR method, and the results showed that the expression amount of AKR1C1 in ECC was highest, whereas AKR1C2 was highly expressed in most tumors, and the difference was not significant (fig. 1C). We therefore speculate that AKR1C1 is specifically highly expressed in ECC.

Finally, we used immunohistochemical methods to detect AKR1C1 expression in ECC tissues and its paracancerous control tissues. The results showed that AKR1C1 was highly expressed in ECC, but hardly expressed in the paracancerous control tissues (see panel D). The immunohistochemical method and conditions were as follows (fozhou mezzo technology development limited immunohistochemical and DAB chromogenic kit):

Dewaxing and hydrating paraffin coated tissue slices, namely roasting the slices at 56 ℃ for 2 hours, dewaxing the slices in xylene for three times for 10 minutes each time, putting the slices into a combination box containing absolute ethyl alcohol, 95% ethyl alcohol and 75% ethyl alcohol in sequence, and washing the slices with running water for 5 minutes each time for 10 minutes each time. And (3) carrying out tissue antigen retrieval by a microwave method, namely heating and boiling the 1 Xcitric acid tissue antigen retrieval liquid, adding the tissue slices during boiling, continuing microwave heating for 4 minutes, and cooling at room temperature.

Taking out tissue slices, removing surface repair liquid, adding 2 drops of peroxidase blocking solution on each tissue slice, incubating for 10 min at room temperature, removing surface blocking solution, washing 3 times with PBS, adding 2 drops of normal non-immune animal serum on each slice, and incubating for 10 min at room temperature. The surface serum of the tissue pieces was removed, 2 drops of AKR1C1 antibody diluted 1:50 were added to each slice, and the slices were placed in a refrigerator and incubated overnight at 4 ℃. The next day, tissue pieces are taken out, washed 3 times by PBS, 2 drops of biotin-labeled secondary antibody are added to each slice, incubated for 10 minutes at room temperature, washed 3 times by PBS, 2 drops of streptomycete biotin-peroxidase solution are added to each tissue piece, incubated for 10 minutes at room temperature, washed 3 times by PBS, 3 drops of freshly prepared DAB solution are added to each slice (300 mu l of PBS is taken, one drop of each reagent in a DAB color development kit is added to each PBS), the tissue pieces are stained for 5-10 minutes, and the staining condition is observed under a microscope. Tap water rinse several times, add 2 drops of hematoxylin on each tissue piece, counterstain for 3 minutes, PBS rinse back to blue. And dehydrating and drying with gradient ethanol (absolute ethanol, 95% ethanol, 75% ethanol) for 5min, and sealing with xylene transparent and neutral resin.

The results show that AKR1C1 is specifically and highly expressed in extrahepatic cholangiocarcinoma, and the expression of AKR1C2-C4 in ECC is not intentional, which suggests that AKR1C1 is very likely to become a diagnostic and therapeutic target of extrahepatic cholangiocarcinoma.

Example 2 AKR1C1 as a diagnostic, metastatic and/or prognostic marker for extrahepatic bile duct cancer

To clarify the role of AKR1C1 in extrahepatic cholangiocarcinoma progression, we collected tumor tissues of 55 patients with initial ECC without radiotherapy, chemotherapy or immunotherapy prior to surgery and their paracancerous control normal tissues, and tumor specimens of extrahepatic cholangiocarcinoma patients were diagnosed as extrahepatic cholangiocarcinoma by pathologists. Tumors were staged (stage I-IV) according to the united states joint cancer committee tumor-lymph node-metastasis (TNM) stage of 2010. The degree of tumor differentiation is defined according to the world health organization standards (hyperdifferentiation, mesodifferentiation and hypodifferentiation). Follow-up was performed on all patients.

The expression of AKR1C1 in the tumor of the patient and the normal tissues of the paracancerous control of the tumor of the patient is detected by adopting an immunohistochemical method, and the correlation of the expression and the clinical pathological parameters of the patient is analyzed. The results showed that AKR1C1 was not substantially expressed in the epithelium of normal bile duct, and the expression level of AKR1C1 was gradually increased with the gradual decrease of the ECC differentiation degree (FIG. 2A), suggesting that AKR1C1 is closely related to the malignancy degree of ECC. Continuing the analysis of the correlation of the pathological parameters of AKR1C1 and ECC (see Table 4), the results show that the expression of AKR1C1 is closely related to the size of primary tumor and lymph node metastasis, and the larger the primary tumor of the patient with high expression of AKR1C1 is, the more easily the lymph node metastasis occurs. Furthermore, kaplan-Meier survival analysis showed that patients with high expression of AKR1C1 had shorter relapse-free and overall survival compared to patients with low expression of AKR1C1 (see fig. 2B). The above results demonstrate that AKR1C1 can be a diagnostic, metastatic and/or prognostic marker for extrahepatic cholangiocarcinoma.

Table 4.55 cases of related clinical data for extrahepatic cholangiocarcinoma patients

*P<0.05,**P<0.01

Example 3 Down-regulating AKR1C1 inhibits proliferation and migration of extrahepatic bile duct cancer cells, promotes death of extrahepatic bile duct cancer cells

To further elucidate the function and mechanism of AKR1C1 in the progression of extrahepatic cholangiocarcinoma, we first constructed the DOX-induced shRNA-AKR1C1 plasmid and performed lentiviral coating. shRNA-AKR C1 target sequence CACCAAAUUGGCAAUUGAA (SEQ ID NO. 11).

ECC cells QBC939 were infected with lentivirus at MOI of 50, puromycin 4ug/ml was added to the blank uninfected virus and virus-infected virus cells 72 hours after virus infection, DOX 5ug/ml was added to the infected virus cells after killing all blank uninfected virus cells to induce shRNa ug-aKR C1 expression, cells were collected 48 hours later, and the efficiency of down-regulating AKR1C1 was examined by RT-PCR and western blot (FIG. 3A), and the results showed that the down-regulating efficiency of AKR1C1 was about 55% -70%. It was demonstrated that DOX-induced shRNA-AKR C1 stable transformants were successfully constructed.

Afterwards, we examined the effect of AKR1C1 on proliferation of ECC cells QBC939 using CCK8 and the colony formation method.

The CCK8 detection method is as follows:

QBC939 stable cells in log phase were digested, centrifuged, resuspended, and incubated overnight in 96-well plates with 5000 cells per well, 100ul of complete medium, 37 ℃ in 5% CO ₂ cell incubator. The DOX addition of 5ug/ml induced shRNA-AKR C1 expression, and after 24 hours, 10 μl of CCK-8 solution was added to the assay wells every 12 hours, and cell proliferation was detected at 450 nm.

The CCK8 assay showed that proliferation rate of QBC939 cells was significantly slowed after down AKR1C1 (fig. 3B), indicating that AKR1C1 overexpression may promote proliferation of QBC939 cells.

The clone formation method is as follows:

The QBC939 stable rotation cells in the logarithmic growth phase are digested, centrifuged and resuspended, the cell suspension is subjected to gradient dilution, and each group of cells is respectively inoculated into 6-well plates at the gradient density of 50, 100 and 200 cells per well of each 6-well plate. The cells were incubated overnight in a 37℃and 5% CO ₂ cell incubator. DOX of 5ug/ml is added to induce shRNA-AKR C1 expression, and the culture is carried out for 2-3 weeks. When macroscopic clones appeared in the wells, the culture was terminated. The supernatant was discarded and carefully rinsed 2 times with PBS. Cells were fixed by adding 1ml of 4% paraformaldehyde to each well for 10 minutes. Then removing the fixing solution, carefully flushing for 2 times by using PBS, adding a proper amount of crystal violet staining solution for dyeing for 10 minutes, and finally, gently flushing by using running water to remove the staining solution, and taking a picture after air drying.

The results of the clone formation experiments showed that down-regulation of AKR1C1 significantly inhibited proliferation of QBC939 cells compared to the control group (fig. 3C), also demonstrating that high AKR1C1 expression can promote proliferation of QBC939 cells.

We continue to examine the effect of AKR1C1 on ECC cell QBC939 death using PI staining and flow cytometry. The procedure for PI staining to detect QBC939 cell death was as follows:

QBC939 stable cells were digested, centrifuged, resuspended, plated in 6-well plates, induced by the addition of 5ug/ml DOX to shRNA-AKR C1 expression, after 48 hours, the medium was discarded from each experimental group and washed once with PBS. Adding appropriate amount of PBS, adding 1 μl of PI dye into each experimental group, incubating in incubator for 5-10 min under dark condition, taking out, photographing with fluorescence microscope, and observing the number of red spots.

PI staining results showed that downregulation of red spots of QBC939 cells in AKR1C1 group was more, suggesting that downregulation of AKR1C1 may promote death of QBC939 cells (fig. 3D).

The flow cytometry detection method is as follows:

the QBC939 stable rotation cells in logarithmic growth phase are digested, centrifuged and resuspended and then spread in a 12-well plate, 5ug/ml DOX is added to induce shRNA-AKR C1 expression, after 48 hours, the collected cells are digested with EDTA-free pancreatin, the cells are resuspended once with precooled 1 XPBS (4 ℃) for 5 minutes at 1200rpm, the cells are washed, 100 μl PBS is used to resuspend the cells, 1 μl SYTOX GREEN DEAD CELL STAIN is added to each tube of cells, the cells are incubated for 20 minutes at room temperature, then 1ml PBS is added for one time, 1000rpm is added for 5 minutes, an appropriate amount of PBS is added for resuspension, and the cells are detected on the machine.

The flow assay showed that down-regulating AKR1C1 group cells died more than the control group, further indicating that down-regulating AKR1C1 can promote QBC939 cell death (fig. 3E).

To clarify the effect of AKR1C1 on ECC cell migration, we used transwell for migration detection, the procedure was as follows:

AKR1C1 in QBC939 cells was down-regulated by the method described above, after which the control and down-regulated groups were resuspended in serum-free medium and plated in transwell cells, 5000 cells per well, and 600 μl of 5% serum-containing medium was added to the 24-well plate in the lower chamber for further incubation for 24 hours. The cells were removed, medium was discarded, and washed twice with PBS. Cells were then fixed with 4% formaldehyde for 10 minutes, formaldehyde was removed and washed twice in PBS. Placing into a new 24-hole plate hole, adding crystal violet staining solution into the upper and lower chambers respectively, standing at room temperature for 15 min, removing crystal violet, washing twice with PBS, slightly wiping off cells on the upper chamber surface with cotton swab, air drying at room temperature, placing on a glass slide, observing cell number with a microscope, and photographing.

Transwell results showed that the number of cell membrane penetrations of AKR1C1 group was significantly lower than that of control group (fig. 3F), suggesting that down-regulation of AKR1C1 could inhibit the migration of extrahepatic bile duct cancer cells.

The results show that AKR1C1 is specifically and highly expressed in extrahepatic cholangiocarcinoma, and the high expression is closely related to the high malignancy degree of ECC patients, and primary tumors of patients with high expression of AKR1C1 are larger and lymph node metastasis is more likely to occur. Meanwhile, compared with patients with low AKR1C1 expression, the patients with high AKR1C1 expression have shorter relapse-free survival time and total survival time. Down-regulating AKR1C1 can inhibit proliferation and migration of extrahepatic bile duct cancer cells, and promote death of extrahepatic bile duct cancer cells. The result suggests that there is a close correlation between AKR1C1 and the occurrence and development of ECC tumors, and thus the AKR1C1 can be used as a marker for tumor diagnosis, tumor treatment scheme selection and tumor prognosis evaluation.

While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.

Claims

1. Application of reagents for detecting AKR1C1 expression in the preparation of diagnostic, staging, grading and prognosis assessment kits for extrahepatic cholangiocarcinoma.

2. The use according to claim 1, characterized in that the reagent for detecting the expression level of AKR1C1 is a reagent for detecting the expression level of AKR1C1 in a biological sample at the gene level and/or protein level; and the kit comprises a reagent for detecting the expression level of AKR1C1 in a biological sample.

3. The use according to claim 2, characterized in that the reagent for detecting the expression level of AKR1C1 in a biological sample is selected from the group consisting of one or more detection techniques or methods: immunohistochemistry, Western blot, and qRT-PCR.

4. The use according to claim 3, characterized in that the reagent for detecting the expression level of AKR1C1 in a biological sample comprises a PCR primer with detection specificity for the AKR1C1 gene, or an antibody that specifically binds to the AKR1C1 protein.

5. The use according to claim 4, characterized in that the PCR primers with detection specificity for the AKR1C1 gene are as shown in SEQ ID NOs. 1 and 2.

6. A kit for diagnosing, staging, grading or evaluating prognosis of extrahepatic bile duct cancer, characterized in that the kit comprises a reagent for detecting the content of AKR1C1 in a biological sample.

7. The kit according to claim 6, characterized in that the kit is composed of a reverse transcription system, a primer system and an amplification system, or is composed of a tumor tissue paraffin section preparation reagent system, an antigen retrieval reagent system and an antibody system, and the primer system includes PCR primers shown as SEQ ID NOs. 1 to 2 and SEQ ID NOs. 9 to 10.

8. Use of a substance that inhibits AKR1C1 in the preparation of a drug for treating extrahepatic bile duct cancer, characterized in that the substance that inhibits AKR1C1 is a shRNA that inhibits AKR1C1 expression or a recombinant expression vector or a transgenic cell line containing the shRNA, and the target sequence of the shRNA is shown in SEQ ID NO.11.

9. A pharmaceutical composition for treating extrahepatic bile duct cancer, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient comprises a shRNA that inhibits AKR1C1 expression or a recombinant expression vector or a transgenic cell line comprising the shRNA, and the target sequence of the shRNA is shown in SEQ ID NO. 11.