CN114107495B

CN114107495B - Use of DUXAP8 in diagnosis, treatment and prevention of endometrial cancer

Info

Publication number: CN114107495B
Application number: CN202111225835.3A
Authority: CN
Inventors: 方军; 袁奕; 范许云; 何胜祥
Original assignee: Anhui Tongke Biotechnology Co ltd
Current assignee: Anhui Tongke Biotechnology Co ltd
Priority date: 2021-10-21
Filing date: 2021-10-21
Publication date: 2023-06-16
Anticipated expiration: 2041-10-21
Also published as: CN114107495A

Abstract

The invention discloses application of DUXAP8 in diagnosis, treatment and prevention of endometrial cancer. In particular to the use of an agent for reducing or inhibiting the expression of DUXAP8 in the preparation of a medicament for the treatment and/or prevention of endometrial cancer. The invention successfully provides a diagnosis and prognosis biomarker for endometrial cancer through detecting the change of gene expression quantity of long-chain non-coding RNA DUXAP 8. By reducing the gene expression level of the DUXAP8, the proliferation and the clone formation of endometrial cancer cells are successfully and effectively reduced, so the invention provides potential application of the DUXAP8 in preparing a pharmaceutical composition for treating endometrial cancer.

Description

Use of DUXAP8 in diagnosis, treatment and prevention of endometrial cancer

Technical Field

The invention belongs to the field of cancers, and particularly relates to application of DUXAP8 in diagnosis, treatment and prevention of endometrial cancer.

Background

Endometrial Cancer (EC) is one of the most common gynaecological tumors worldwide. In China, the incidence rate is inferior to cervical cancer and ovarian cancer, and is in the 3 rd position of gynecological malignant tumor. In recent years, the incidence rate thereof has been increasing. Despite great progress in diagnostic techniques, surgical treatments, chemotherapy and radiation therapy, the increase in survival rate of endometrial cancer patients is still not desirable. Therefore, the exact mechanism of endometrial cancer occurrence, development and metastasis is explored and clarified on a molecular level, and is important for searching new drug targets and screening effective early diagnosis molecular markers.

With the widespread transcription of the human genome, most human transcriptomes consist of non-coding RNAs (ncrnas). Non-coding RNAs lack protein-coding ability, and their main role is to regulate transcription and expression of coding genes to affect the growth and development of organisms, differentiation, proliferation, apoptosis and stress of cells. Long non-coding RNAs (lncRNAs) are defined as transcripts of more than 200 nucleotides depending on transcript size. They affect the expression of target genes by interacting with other molecules (such as DNA, RNA, and proteins) to act as modulators in disease progression or interacting with target RNAs (e.g., mRNA and microRNA (miRNA)) or specific signaling proteins to modulate related signaling pathways. Previous studies have shown that lncRNA is differentially expressed in endometrial cancer tissue, and altering its expression level can inhibit proliferation of endometrial cancer cells.

In the study of endometrial cancer, several lncRNA expression patterns, clinical relevance, biological function and intrinsic mechanisms have been discovered. For example, lncRNA NEAT1 promotes endometrial cancer cell proliferation, migration and invasion by modulating miR-144-3p/EZH2, up-regulating the expression of EZH 2. LncRNA TDRG1 enhances the tumorigenicity of endometrial cancer by binding and targeting VEGF-Sub>A protein. Although a small fraction of lncRNAs have been studied in endometrial cancer at present, current research on lncRNA molecules in endometrial cancer regulation mechanisms is still in the onset phase, and the clinical significance and potential function of most lncRNAs in endometrial carcinogenesis and progression is still unclear. Therefore, by researching the occurrence and development mechanism of more lncRNA in endometrial cancer, the method plays an important role in diagnosis, prevention and treatment of endometrial cancer and provides a potential target point for targeted treatment of endometrial cancer.

Disclosure of Invention

In order to perfect the defects of the existing technology for diagnosing and treating endometrial cancer, the invention provides an application of an agent for reducing or inhibiting the expression of DUXAP8 in preparing a medicament for treating and/or preventing endometrial cancer, an application of an agent for detecting the expression level of DUXAP8 in preparing a product for diagnosing endometrial cancer and a method for screening medicaments for resisting endometrial cancer. The invention takes long-chain non-coding RNA DUXAP8 as a diagnosis and prognosis biomarker of endometrial cancer, and inhibits malignant proliferation and clone formation of endometrial cancer cells by reducing the gene expression quantity of the DUXAP8, thereby providing a new target for targeted therapy of endometrial cancer.

There is currently no report on DUXAP8 in endometrial cancer.

1. According to the study, through a TCGA database, the expression data of DUXAP8 in tumor tissues of 524 endometrial cancer patients and 23 control normal tissues are obtained, and through analysis, compared with the control normal tissues, the DUXAP8 is remarkably and highly expressed in the endometrial cancer tumor tissues, namely the DUXAP8 can be used as an endometrial cancer diagnosis marker.

2. Furthermore, in the cancer tissues of 524 cases of endometrial cancer patients, the expression quantity of DUXAP8 in the tumor tissues of the low differentiation group is obviously higher than that of the tumor tissues of the high differentiation group;

3. subsequently, 524 endometrial cancer patients were grouped by DUXAP8 expression level, and the total survival time of the low-expression DUXAP8 group of endometrial cancer patients was found to be significantly longer than that of the high-expression DUXAP8 group, i.e., DUXAP8 was used as a prognostic marker for endometrial cancer.

4. In vitro experiments show that compared with the Ishikawa cell strain of the highly differentiated endometrial cancer, the expression level of DUXAP8 in the poorly differentiated KLE cells is remarkably increased, which is consistent with the analysis result of the TCGA database.

5. The invention also provides shRNA for inhibiting human DUXAP8 gene expression and application thereof. The invention screens shRNA with high transfection efficiency and high interference efficiency, constructs a nucleic acid construct and a slow virus containing the shRNA sequence, and can efficiently inhibit the expression of DUXAP8 genes in human cells.

6. Furthermore, the expression level of DUXAP8 in the edited endometrial cancer cells is obviously reduced, so that the proliferation and tumor stem of the endometrial cancer cells can be effectively inhibited, and a new direction is opened for the treatment of endometrial cancer.

In a first aspect the invention provides the use of an agent that reduces or inhibits expression of DUXAP8 in the manufacture of a medicament for the treatment and/or prophylaxis of endometrial cancer.

In such applications, the agent is preferably a shRNA targeting DUXAP 8.

In a preferred embodiment, the sense and antisense strands of the shRNA comprise the sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5, respectively.

The shRNA is preferably present in an expression vector.

The expression vector is preferably a lentiviral expression vector.

In the present invention, the expression vectors are, for example, pLKO.1-puro, pRNAT-U6.1/neo, pSilencer2.1-U6 neo, pSilencer 4.1.1-CMV puro, pLVX-shRNA1 and pAAVtdTomato-shRNA.

In a preferred embodiment, the lentiviral expression vector is pLKO.1-puro.

The expression vector is preferably present in a virus.

The medicament preferably further comprises a delivery vehicle for delivering an expression vector containing the shRNA into a cell, such as a viral delivery vehicle exemplified by lentiviral vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and a liposoluble vehicle exemplified by liposomes.

In such applications, the agent is preferably the only active ingredient or one of the active ingredients of the medicament.

In a second aspect, the invention provides the use of an agent for detecting the expression level of DUXAP8 in the manufacture of a product for diagnosing endometrial cancer.

The agent for detecting the expression level of DUXAP8 is preferably a specific probe for DUXAP8 (e.g. with a detection label, usually complementary to the gene of interest), a gene chip or PCR primer.

In a preferred embodiment, the analysis of the expression level of DUXAP8 is by qRT-PCR.

In a preferred embodiment, the PCR primers comprise the sequences shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively.

In a third aspect, the present invention provides a method of screening for an anti-endometrial cancer agent, said method comprising the steps of:

(1) Determining the expression level of DUXAP8 in endometrial cancer cells;

(2) Contacting the candidate agent with the endometrial cancer cells of step (1) and determining the expression level of DUXAP8 after the contacting;

(3) If the expression level of DUXAP8 in step (2) is significantly reduced, it is indicated that the drug candidate has potential against endometrial cancer cells.

In the method, the time of the contact in step (2) is preferably 24 to 72 hours.

In the method, the candidate drug is preferably used at a concentration of 1nM to 50. Mu.M.

In the present invention, significant means statistically different in the art, for example, P value less than 0.05, P value less than 0.01.

In the invention, the Ensembl database of DUXAP8 is numbered as ENSG00000271672, and the sequence is shown as SEQ ID NO. 14.

The invention has the positive progress effects that:

the invention provides application of long-chain non-coding RNA DUXAP8 serving as a biomarker in diagnosis, treatment and prevention of endometrial cancer. By detecting the change of the gene expression quantity of the long-chain non-coding RNA DUXAP8, the method successfully provides a diagnosis and prognosis biomarker which can be used as endometrial cancer. By reducing the gene expression level of the DUXAP8, the proliferation and the clone formation of endometrial cancer cells are successfully and effectively reduced, so the invention provides potential application of the DUXAP8 in preparing a pharmaceutical composition for treating endometrial cancer.

Drawings

FIG. 1 shows a differential expression pattern of the gene DUXAP8 in cancer tissues of patients with endometrial cancer and normal control tissues.

FIG. 2 shows a graph of differential expression of the gene DUXAP8 in endometrial cancer tissue of varying degrees of differentiation.

FIG. 3 shows an analysis of the prognostic relevance of the gene DUXAP8 to endometrial cancer patients.

FIG. 4 shows a graph of differential expression of the gene DUXAP8 in endometrial cancer cell lines of varying degrees of differentiation.

Figure 5 shows a validation of detection of lentiviral mediated shRNA inhibition of DUXAP8 gene in endometrial cancer cells.

FIG. 6 shows a graph of the effect of detecting DUXAP8 on endometrial cancer cell proliferation using CCK 8.

FIG. 7 shows a graph of the effect of detecting the DUXAP8 gene on endometrial cancer cell tumor stem.

FIG. 8 shows analysis of expression of DUXAP8, lncRNA LOXL1-AS1, lncRNA PCAT1 in cancer tissue of endometrial cancer patients and normal control tissue.

FIG. 9 shows a graph of the prognosis correlation of DUXAP8, lncRNA LOXL1-AS1, lncRNA PCAT1 and endometrial cancer patients.

Detailed Description

EXAMPLE 1 differential expression of the DuXAP8 Gene in endometrial cancer tissue, normal control tissue, and prognosis-related analysis

1. Using TCGA (The Cancer Genome Atlas) database, the high-throughput sequencing results of 524 endometrial cancer patients' cancer tissues and 23 normal controls were analyzed to obtain the differential endometrial cancer gene expression profile, wherein the gene DUXAP8 was significantly highly expressed in endometrial cancer tissues (fig. 1).

2. Further, the expression level of DUXAP8 in cancer tissue and the differentiation degree of endometrial cancer tissue of 524 cases of endometrial cancer patients were analyzed against TCGA database, wherein the expression level of DUXAP8 in cancer tissue of the low differentiation group was significantly higher than that of the high differentiation group (fig. 2).

3. Further, by performing a correlation analysis of the cancer tissue DUXAP8 expression level and the prognosis of the patients in 524 cases of endometrial cancer patients against the TCGA database, the groups of 524 cases of endometrial cancer patients were grouped by the expression level of DUXAP8, and the total survival time of the low-expression DUXAP8 group of endometrial cancer patients was found to be significantly longer than that of the high-expression DUXAP8 group, and the DUXAP8 was significantly negatively correlated with the prognosis of the endometrial cancer patients (fig. 3).

Example 2: the DUXAP8 gene is used as a diagnosis and prognosis biomarker of endometrial cancer and is effectively compared with other lncRNA in the prior art

Literature (PMID: 32006897), literature (PMID: 31136293; PMID: 31927050) report lncRNA LOXL1-AS1 (Ensembl database No. ENSG 00000261801), lncRNA PCAT1 (Ensembl database No. ENSG 00000253438), respectively, AS diagnostic and prognostic markers for endometrial cancer. The inventors compared DUXAP8 of the present application with lncRNA LOXL1-AS1, lncRNA PCAT1 with respect to endometrial cancer diagnosis and prognosis effects.

1. Using GEPIA2 (Gene Expression Profiling Interactive Analysis 2) database (http:// GEPIA2.Cancer-pku. Cn/# index), 174 endometrial cancer patient cancer tissues and 91 normal control high-throughput sequencing results were obtained, and the expression conditions of DUXAP8, lncRNA LOXL1-AS1, lncRNA PCAT1 in endometrial cancer patient cancer tissues and normal control tissues were analyzed, respectively, to detect the diagnostic effect of the genes on endometrial cancer. Wherein the gene DUXAP8 was significantly highly expressed in endometrial cancer tissue compared to normal control tissue, whereas lncRNA LOXL1-AS1, lncRNA PCAT1 were expressed without statistical differences in endometrial cancer tissue (fig. 8). Therefore, the DUXAP8 gene has better diagnosis effect on endometrial cancer than the known reported lncRNA LOXL1-AS1 and lncRNA PCAT1.

2. Further, according to correlation analysis of cancer tissue DUXAP8, lncRNA LOXL1-AS1 and lncRNA PCAT1 expression levels of 174 endometrial cancer patients in a GEPIA2 database and prognosis of the patients, 174 endometrial cancer patients are respectively grouped according to the DUXAP8, lncRNA LOXL1-AS1 and lncRNA PCAT1 expression levels, the total survival time of the low-expression DUXAP8 group endometrial cancer patients is obviously longer than that of the high-expression DUXAP8 group, and the DUXAP8 and the prognosis of the endometrial cancer patients are obviously and negatively correlated; while lncRNA LOXL1-AS1, lncRNA PCAT1 had no significant correlation with endometrial cancer patient prognosis (fig. 9). Therefore, the effect of the DUXAP8 gene of the patent on prognosis judgment of endometrial cancer is superior to that of the known reported lncRNA LOXL1-AS1 and lncRNA PCAT1.

In summary, the DUXAP8 gene is used AS a biomarker for diagnosing and prognosing endometrial cancer, and the effect is superior to that of lncRNA LOXL1-AS1 and lncRNA PCAT1 in the prior art.

Example 3 differential expression of the DuXAP8 Gene in endometrial cancer cell lines of varying degrees of differentiation

1. Cell culture

Human endometrial cancer cell lines Ishikawa (hyperdifferentiation) and KLE (hypodifferentiation) were cultured using DMEM medium containing 10% fetal bovine serum and 1% diabody at 37deg.C, 5% CO ₂ The relative humidity is 90%Is cultured in an incubator of (a). The solution was changed 1 time from 2 to 3 days and passaged by routine digestion with 0.25% trypsin containing EDTA. Human endometrial cancer cell lines Ishikawa (high differentiation) and KLE (low differentiation) were both purchased from the marsupenario life technologies limited company.

2. RNA extraction

The cultured cells were collected and Total RNA of the cells was extracted according to the Total RNA extraction kit (Tiangen Biochemical Co., ltd.).

3. Reverse transcription

cDNA was synthesized by reverse transcription using a reverse transcription kit (Shanghai Co., ltd.).

4. qPCR amplification assay

The reaction procedure is: the mixture was pre-denatured at 95℃for 10min, then denatured at 95℃for 15s, annealed at 60℃for 30s, and extended at 72℃for 30s, and the total was subjected to 38 cycles. The ABI 7500 fluorescent quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescent signals during the ramp up process to obtain a melting curve. Real-Time PCR Using 2 ^-△△Ct The method is used for relative quantitative analysis. Primer sequences are shown in Table 1:

TABLE 1 specific amplification primer sequences and related information

The results are shown in FIG. 4, where the DUXAP8 gene was up-regulated in the poorly differentiated endometrial cancer cell line (KLE) compared to the highly differentiated endometrial cancer cell line (Ishikawa), the differences being statistically significant (P < 0.05).

EXAMPLE 4 construction of shRNA lentiviral expression vector of DUXAP8 Gene

1. shRNA target design

(1) The Ensembl database inquires mRNA information (the sequence is shown as SEQ ID NO: 14) of a DUXAP8 (ENSG 00000271672) gene, and a plurality of 19-21 nt RNA interference target sequences are designed according to the shRNA design principle. After evaluation and measurement by designed software, the following sequences are selected as interference targets:

Human-DUXAP8-1：GGAACTTCCCAAACCTCCATGATTT，SEQ ID NO:1；

Human-DUXAP8-2：AAGATAAAGGTGGTTTCCACAAGAA，SEQ ID NO:2；

Human-DUXAP8-3：CAGCATACTTCAAATTCACAGCAAA，SEQ ID NO:3。

2. DNA oligo sequence Synthesis

And designing a shRNA interference sequence according to the selected target sequence, and adding proper restriction enzyme cutting sites at two ends to complete the construction of the vector. In addition, TTTTT or TTTTTT transcription termination signals are added at the 3 '-end of the forward strand, and termination signal complementary sequences are added at the 5' -end of the reverse strand; c is added at the 5 'end of the positive strand, complementary bases are added at the 3' end of the negative strand, and a stem-loop sequence (CTCGAG) is positioned in the center of the oligonucleotides and connected with two oligonucleotides; 3 interfering sequences were designed, and a missense sequence was designed as a control group, and single-stranded DNA oligo was synthesized by Souzhou Jin Weizhi Biotechnology Co.

The shRNA interference sequence is as follows:

the sense strand of DUXAP 8-shRNA-1:

CCGGGGAACTTCCCAAACCTCCATGATTTCTCGAGAAATCATGGAGGTTTGGGAAGTTCCTTTTTG，SEQ ID NO:4；

DUXAP8-shRNA-1 antisense strand:

AATTCAAAAAGGAACTTCCCAAACCTCCATGATTTCTCGAGAAATCATGGAGGTTTGGGAAGTTCC，SEQ ID NO:5；

DUXAP8-shRNA-2 sense strand:

CCGGAAGATAAAGGTGGTTTCCACAAGAACTCGAGTTCTTGTGGAAACCACCTTTATCTTTTTTTG，SEQ ID NO:6；

DUXAP8-shRNA-2 antisense strand:

AATTCAAAAAAAGATAAAGGTGGTTTCCACAAGAACTCGAGTTCTTGTGGAAACCACCTTTATCTT，SEQ ID NO:7；

DUXAP8-shRNA-3 sense strand:

CCGGCAGCATACTTCAAATTCACAGCAAACTCGAGTTTGCTGTGAATTTGAAGTATGCTGTTTTTG，SEQ ID NO:8；

DUXAP8-shRNA-3 antisense strand:

AATTCAAAAACAGCATACTTCAAATTCACAGCAAACTCGAGTTTGCTGTGAATTTGAAGTATGCTG，SEQ ID NO:9；

3. construction of shRNA lentiviral expression vector of DUXAP8 Gene

(1) Annealing of DNA oligo sequence

Mixing sense strand (100. Mu.M) and antisense strand (100. Mu.M) DNA oligo1:1 uniformly to make its final concentration be 50. Mu.M, adding annealing buffer solution, and annealing (95 deg.C, 30s;72 deg.C, 2min;37 deg.C, 2min;25 deg.C, 2 min) on PCR instrument to form DNA double strand, namely shRNA template. The annealed shRNA template was diluted with RNase-free water to a final concentration of 200nM for ligation reaction.

(2) Carrier linearization

The lentiviral expression vector pLKO.1-puro (Biovector plasmid vector cell Gene Collection) was subjected to double digestion with AgeI/EcoRI, and the vector was linearized, and the digestion system was as shown in Table 2:

TABLE 2 enzyme digestion system

Component (A)	Dosage of
		Vector	4μg
AgeI	1uL
		EcoRI	1uL
Buffer(10×)	5uL
		ddH ₂ O	Make up to 50 mu L

The resulting vector was subjected to 1% agarose gel electrophoresis at 37℃for 3 hours, and the desired fragment was recovered and diluted to 50 ng/. Mu.L with RNase-free water.

(3) Ligation of the fragment of interest with the vector

And (3) connecting the linearized pLKO.1-puro obtained in the step (2) with the shRNA template obtained in the step (1), and carrying out a connection reaction by adopting T4 ligase. The connection system is shown in Table 3:

TABLE 3 connection System

Component (A)	Dosage of
		Linearization Vector	1uL
Oligonucleotide fragments	1uL
		T4 DNA ligase	1uL
T4 Buffer(10×)	2uL
		ddH ₂ O	15μL

The ligation was carried out overnight at 16℃after which the transformation experiment was carried out.

(4) PCR identification and sequencing of transformed and positive clones

Adding 5 μl of the ligation product into 50 μl of E.coli competent cells, ice-bathing for 30min, heat-shocking at 42deg.C for 90s, and ice-bathing for 2min; adding 500 mu L of LB liquid medium without antibiotics, and shake culturing at 200rpm at 37 ℃ for 1h; the bacterial liquid is evenly smeared on LB solid medium containing Amp, and cultured overnight in a 37 ℃ incubator. The next day, single colonies are selected, bacterial liquid PCR identification is carried out, positive clones are selected for sequencing (Jin Weizhi Biotechnology Co., st.) and are subjected to sequencing comparison, and the positive clones are identified to be the successfully constructed pLKO.1-puro-DUXAP8-shRNA lentiviral vector.

(5) Lentivirus packaging and titer determination

HEK-293T cells (Wohunorace life technologies Co., ltd.) were co-transfected with the extracted recombinant plasmid pLKO.1-puro-DUXAP8-shRNA, helper plasmid psPAX2 and PMD2.G (Biovector plasmid vector cell Gene Collection), virus supernatants were collected at 48h and 72h, respectively, cell morphology and green fluorescent protein GFP expression were observed, the extracted viruses were purified and concentrated, virus titer was measured by a gradient dilution method, and the prepared virus concentrate was packaged at-80℃for preservation.

Acquisition of the Scramble virus: the pLKO.1-puro plasmid containing the missense sequence, the auxiliary plasmid psPAX2 and the PMD2.G are transfected into HEK-293T cells together, virus supernatants are collected respectively for 48 hours and 72 hours, the cell morphology and green fluorescent protein GFP expression are observed, the extracted viruses are purified and concentrated, the virus titer is measured by a gradient dilution method, and the prepared virus concentrate is split-packed at the temperature of minus 80 ℃ for preservation.

EXAMPLE 5 DUXAP8-shRNA lentivirus infection of human endometrial cancer cells and stable Trans-strain establishment

(1) Taking Ishikawa cells in logarithmic growth phase, adjusting cell concentration to 2×10 ⁵ Well inoculated in 6-well plate;

(2) The next day, adding DUXAP8-shRNA1, DUXAP8-shRNA2, DUXAP8-shRNA3 and a Scramble virus solution into a culture medium respectively, adding 8 mug of transfection enhancing reagent polybrene into each hole, shaking and mixing uniformly;

(3) Culturing for 12 hours, replacing a culture medium containing virus diluent, and repeating the same method for 2 times; fluorescence and infection efficiency were observed under a microscope. If the fluorescence efficiency is more than 80%, the cell state is good, and the cell fusion degree is more than 80%, the infection is successful.

Thereafter, the culture medium containing puromycin (concentration 1. Mu.g/ml) was used for the selection culture. The culture solution is replaced every 72 hours, and stable transfer cell lines can be obtained 3 times continuously.

Example 6 DUXAP8-shRNA lentiviral interference efficiency validation

(1) Experimental grouping: an infected negative control group (Scramble group), an infected DUXAP8-shRNA-1 group (shRNA-1 group), an infected DUXAP8-shRNA-2 group (shRNA-2 group), an infected DUXAP8-shRNA-3 group (shRNA-3 group).

(2) The Total RNA of the cells was extracted from each cell group and the Control group cells cultured in synchronization according to the Total RNA extraction kit (Tiangen Biochemical technology Co., ltd.).

(3) cDNA was synthesized by reverse transcription using a reverse transcription kit (Shanghai Co., ltd.).

(4) Real-Time PCR was performed to detect DUXAP8 cDNA, with the following procedure: the mixture was pre-denatured at 95℃for 10min, then denatured at 95℃for 15s, annealed at 60℃for 30s, and extended at 72℃for 30s, and the total was subjected to 38 cycles. The ABI 7500 fluorescent quantitative PCR instrument selects a melting curve program, and continuously collects sample fluorescent signals during the ramp up process to obtain a melting curve. Real-Time PCR was performed using the 2-DeltaCt method for relative quantitative analysis. The primer sequences are shown in Table 1.

As shown in FIG. 5, the experimental groups DUXAP8-shRNA-1 and DUXAP8-shRNA-3 were able to reduce the expression level of DUXAP8 compared to the Scramble group, wherein the DUXAP8-shRNA-1 group inhibited more significantly with statistical differences, so that DUXAP8-shRNA-1 was used for the subsequent experiments.

Example 7 DUXAP8 Low expression inhibited proliferation of endometrial cancer cells

(1) Experimental grouping: the negative control group (Scramble group) was infected, and the DUXAP8-shRNA-1 group (shRNA-1 group) was infected.

(2) Taking the above groups of logarithmic phase cells, adjusting cell concentration to 3×10 ³ Wells/well were seeded in 96-well plates, 3 multiple wells per group.

(3) The incubation was continued for 1-4 days and the effect of DUXAP8 on cell proliferation was examined using the CCK-8 assay.

As shown in fig. 6, interfering with DUXAP8 expression inhibits the proliferation capacity of endometrial cancer cell lines.

Example 8 DUXAP8 Low expression inhibited tumor stem of endometrial cancer cells

(2) Taking the above groups of logarithmic phase cells, adjusting cell concentration to 5×10 ² The wells were seeded in 6-well plates, 3 multiple wells per group.

(3) The incubation was continued for 10-14 days and the effect of DUXAP8 on tumor dryness was examined using a plate cloning experiment.

As shown in fig. 7, interfering with DUXAP8 expression inhibits the clonogenic capacity of endometrial cancer cell lines, i.e., tumor stem.

SEQUENCE LISTING

<110> Anhui Cooperation Biotech Co., ltd

<120> use of DUXAP8 in diagnosis, treatment and prevention of endometrial cancer

<130> P21017489C

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> human-DUXAP 8-1

<400> 1

ggaacttccc aaacctccat gattt 25

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> human-DUXAP 8-2

<400> 2

aagataaagg tggtttccac aagaa 25

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> human-DUXAP 8-3

<400> 3

cagcatactt caaattcaca gcaaa 25

<210> 4

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8-shRNA-1 sense strand

<400> 4

ccggggaact tcccaaacct ccatgatttc tcgagaaatc atggaggttt gggaagttcc 60

tttttg 66

<210> 5

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8-shRNA-1 antisense strand

<400> 5

aattcaaaaa ggaacttccc aaacctccat gatttctcga gaaatcatgg aggtttggga 60

agttcc 66

<210> 6

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8-shRNA-2 sense strand

<400> 6

ccggaagata aaggtggttt ccacaagaac tcgagttctt gtggaaacca cctttatctt 60

tttttg 66

<210> 7

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8-shRNA-2 antisense strand

<400> 7

aattcaaaaa aagataaagg tggtttccac aagaactcga gttcttgtgg aaaccacctt 60

tatctt 66

<210> 8

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8-shRNA-3 sense strand

<400> 8

ccggcagcat acttcaaatt cacagcaaac tcgagtttgc tgtgaatttg aagtatgctg 60

tttttg 66

<210> 9

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8-shRNA-3 antisense strand

<400> 9

aattcaaaaa cagcatactt caaattcaca gcaaactcga gtttgctgtg aatttgaagt 60

atgctg 66

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8 upstream primer

<400> 10

cactgattcc ttctgagact 20

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8 downstream primer

<400> 11

gagccatact gttgaacct 19

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH upstream primer

<400> 12

caatgacccc ttcattgacc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH downstream primer

<400> 13

gacaagcttc ccgttctcag 20

<210> 14

<211> 2107

<212> DNA

<213> Artificial Sequence

<220>

<223> DUXAP8

<400> 14

acttcctgcg aggcccctgc agcagcagcg gcgtggtcag agcgagcttc ggagaagcag 60

tggtgggttc catgtgatgg tggagtagga ggcaggtctc cgcggttcat ctgtgttgct 120

ctaaatgaca ctgcttcatt attttgatgg ctggagaata tttcctagtg tatgtatatg 180

agagtttctt gatctcttta tctgtggatg aacaggctag tcaccctgct gtggaataga 240

aggccagaat tgatcagtct catctgagag taactttgta cccatcactg attccttctg 300

agactgcctc cacttcccca gcagcctctg gtttcttcat gtggctgcag atggcaggat 360

ttcccaaagg tttctggctg aaacatattc cgtggtgtat ctgtacagca gtttcctcat 420

ccctgcagct gtgtttgaac aggtcattta ccatgctgtc ctccaggttc aacagtatgg 480

ctccaaatga tgaaatttca ttctgatttt ctggctgaag actattctct ttgtgtatgt 540

ccaccacagt tactttatcc cttcatctgt ggatgggcag gatggagtct cgctgtattg 600

cccaggctgg agtgcagtgg catgatctca gctcactgca agctctgctt cctgggttca 660

cgccattctc ctgcctcagc ctcctgagta gctgggatta caggcacccg ccaccacgcc 720

caggaaagaa aaaagaagaa aacaaacctc catacgagaa tgggtctaaa ggaacttccc 780

aaacctccat gattttgcag gaaacaagat aaaggtggtt tccacaagaa aaatggcaca 840

atgtttctca gaagacaatt acataagaat cagcatactt caaattcaca gcaaataatc 900

agacaattga tgaaaatact tacccaaaca ctaattgtag actatgcctt ctgaatatgt 960

ttgtcataaa cttggagtaa ggaatcctca caggcactgg acaattcaaa aaacgtaaag 1020

ttgtttgtta gaatactggt gcttttggat agaaaccctc atccatatcc tggtaaggct 1080

tgaagttgca caggagtttt catttgtcaa aacccagaaa accataagct ttagatttgt 1140

gaattttata ttgtattata tgtgaccttt ctttttaaaa aatgagctgt aagcagtctc 1200

ccagacagta gctcagcctc cagaactctc tttctgcata gttgaagacc cctcttcaca 1260

caagatggta gcaacaaatc ataggtgcaa ttgcaccaaa ttcacagaag atcaattgaa 1320

aatcctcatc aataccttca ctcaaaaacc ttacccaggt tatgctacca aacaaaaact 1380

tgctttagca atcaatgcag aagagtccag aatccagatt tggtttcaga atcaaagagc 1440

taggcatgga ttccagaaaa caccagaacc tgactttaga tttaagccac agccatggac 1500

aagattaacc tggtgtggag tttcaaaata gagaagccag atggtgttgt accacctata 1560

gcacctttca attacacaca gtcatccatg catttatgaa aaacccatac cctgggattg 1620

attccagaga acaacttgct gaagaaattg gtgcttcaga gtcaagagtc caaatttggt 1680

tccaaaatca aagatctaga tttcatctcc agagaaaaag agaacctgtt atgtccttag 1740

aatgagaaga ccagagaaga ccaggggcaa ggtttctgag ggacttcaag gtacagaaga 1800

tacacaaagt ggcaccagcc tcactagcac tctcatttct caagagccag aacatggtga 1860

atacagtcaa gttcagtgta tttgataata tcaatttggg ccccaaatct ctctcacagt 1920

cttcctggga gtctattctt cttccaaaag tgcaagctaa gccttctgaa gatggtaaag 1980

aacttggccg ggtgtggtgg ctcatgcctg taatcccagc actttaggag gctgaggctg 2040

gaagatggct tgagcctagg agtttgaaac cagtctgagc aacatagtaa gaccctgtct 2100

ctattct 2107

<210> 15

<211> 1386

<212> DNA

<213> Artificial Sequence

<220>

<223> GAPDH

<400> 15

gtccggatgc tgcgcctgcg gtagagcggc cgccatgttg caaccgggaa ggaaatgaat 60

gggcagccgt taggaaagcc tgccggtgac taaccctgcg ctcctgcctc gatgggtgga 120

gtcgcgtgtg gcggggaagt caggtggagc gaggctagct ggcccgattt ctcctccggg 180

tgatgctttt cctagattat tctctgattt ggtcgtattg ggcgcctggt caccagggct 240

gcttttaact ctggtaaagt ggatattgtt gccatcaatg accccttcat tgacctcaac 300

tacatggttt acatgttcca atatgattcc acccatggca aattccatgg caccgtcaag 360

gctgagaacg ggaagcttgt catcaatgga aatcccatca ccatcttcca ggagcgagat 420

ccctccaaaa tcaagtgggg cgatgctggc gctgagtacg tcgtggagtc cactggcgtc 480

ttcaccacca tggagaaggc tggggctcat ttgcaggggg gagccaaaag ggtcatcatc 540

tctgccccct ctgctgatgc ccccatgttc gtcatgggtg tgaaccatga gaagtatgac 600

aacagcctca agatcatcag caatgcctcc tgcaccacca actgcttagc acccctggcc 660

aaggtcatcc atgacaactt tggtatcgtg gaaggactca tgaccacagt ccatgccatc 720

actgccaccc agaagactgt ggatggcccc tccgggaaac tgtggcgtga tggccgcggg 780

gctctccaga acatcatccc tgcctctact ggcgctgcca aggctgtggg caaggtcatc 840

cctgagctga acgggaagct cactggcatg gccttccgtg tccccactgc caacgtgtca 900

gtggtggacc tgacctgccg tctagaaaaa cctgccaaat atgatgacat caagaaggtg 960

gtgaagcagg cgtcggaggg ccccctcaag ggcatcctgg gctacactga gcaccaggtg 1020

gtctcctctg acttcaacag cgacacccac tcctccacct ttgacgctgg ggctggcatt 1080

gccctcaacg accactttgt caagctcatt tcctggtatg acaacgaatt tggctacagc 1140

aacagggtgg tggacctcat ggcccacatg gcctccaagg agtaagaccc ctggaccacc 1200

agccccagca agagcacaag aggaagagag agaccctcac tgctggggag tccctgccac 1260

actcagtccc ccaccacact gaatctcccc tcctcacagt tgccatgtag accccttgaa 1320

gaggggaggg gcctagggag ccgcaccttg tcatgtacca tcaataaagt accctgtgct 1380

caacca 1386

Claims

1. Use of an agent that inhibits DUXAP8 expression in the manufacture of a medicament for treating endometrial cancer, wherein the agent is a shRNA that targets DUXAP8, and the sense strand and the antisense strand of the shRNA are respectively the sequences shown as SEQ ID No. 4 and SEQ ID No. 5.

2. The use of claim 1, wherein the shRNA is present in an expression vector.

3. The use according to claim 2, wherein the expression vector is a lentiviral expression vector.

4. The use according to claim 3, wherein the lentiviral expression vector is plko.1-puro.

5. The use according to any one of claims 1 to 4, wherein the agent is the sole active ingredient or one of the active ingredients of the medicament.

6. Use of an agent for detecting DUXAP8 expression levels in the manufacture of a product for diagnosing endometrial cancer.

7. The use according to claim 6, wherein the reagent for detecting the expression level of DUXAP8 is a specific probe, a gene chip or a PCR primer for DUXAP 8.

8. The use according to claim 6 or 7, wherein the analysis of the expression level of DUXAP8 is by means of qRT-PCR.

9. The use according to claim 8, wherein the PCR primers are the sequences shown in SEQ ID NO. 10 and SEQ ID NO. 11, respectively.