CN116356028A - Application of Rad17 as cervical cancer marker and therapeutic target - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and discloses application of Rad17 as a cervical cancer marker and a treatment target. According to the invention, through researches, a novel cervical cancer diagnosis marker Rad17 is provided, occurrence and development of cervical cancer can be clearly represented, the marker presents a specific high expression phenomenon in cervical cancer tissues of clinical patients compared with corresponding normal cervical tissues, and also presents high expression in a human cervical cancer cell line; and it was demonstrated that knocking down Rad17 suppresses cervical cancer malignancy. Rad17 is used as a diagnosis marker of cervical cancer, provides a new molecular target for the treatment of cervical cancer, and provides a new idea for the treatment of cervical cancer.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of Rad17 as a cervical cancer marker and a treatment target.
Background
Cervical cancer is the fourth most common cancer in female cancers in incidence and mortality worldwide, next to breast, colorectal and lung cancers. Persistent high-risk HPV infection is a major factor in cervical cancer occurrence, but its mechanism is not clear. Current HPV vaccines are only able to prevent a few HPV infections and have no therapeutic effect on already infected patient vaccines. How to reduce the incidence of cervical cancer remains an important issue to be addressed. Therefore, the molecular mechanism for developing cervical cancer is of great significance for developing new diagnostic markers and drug targets.
Pap smear is a way to find cervical precancerous lesions and early cervical cancer clinically at present, and the method is used for carrying out primary screening on cervical exfoliated cells, and has good specificity, but poor sensitivity. HPV detection can aid diagnosis, but HPV infection is only a high risk factor and cannot determine whether to progress to the cancer stage. The colposcopy can further detect suspected cervical cancer, but the method has high professional requirements on operators, and is difficult to popularize in areas with lagged medical level in China. Therefore, it is important to find new molecular markers for tumor diagnosis and therapeutic targets.
Cell cycle checkpoints (checkpoints) are some control points in the course of the cell cycle, mainly when DNA is damaged (sometimes including adverse conditions such as nutrient starvation) that provide the cell with sufficient time to repair the damaged DNA (or to pass crisis) by slowing or temporarily suspending the progression of the cell cycle. Cell cycle checkpoint related protein Rad17 is a key detection protein in the process of cell response DNA damage and replication fork blocking signal transduction, and plays a very important role in DNA damage and DNA replication detection. There is no report on the correlation of Rad17 with cervical cancer.
Disclosure of Invention
The object of the present invention is to address the above problems and to provide a new application of Rad17 as a marker for cervical cancer: use of a substance for detecting the expression level of a biomarker, said biomarker being Rad17, in the manufacture of a product for diagnosing cervical cancer.
In the above application protocol, the level of Rad17 gene or protein expression in cervical tissue is detected for an individual suffering from or suspected of suffering from cervical cancer.
In the above application technical scheme, if the Rad17 gene or protein expression level in the diseased cervical tissue of the individual exhibits a specific high expression compared with the normal cervical tissue, the individual is indicated to be a cervical cancer high risk patient.
In the application technical scheme, the product is a reagent or a kit.
In the above application, the substance is used for detecting the protein expression level of the Rad17 gene or the mRNA expression level of the Rad17 gene.
The invention also provides application of the Rad17 serving as a target in screening/preparing a medicament for treating cervical cancer, and the medicament can inhibit the expression of the Rad17 gene and inhibit the malignant characteristics of cervical cancer.
In the application technical scheme, the expression of Rad17 is inhibited, so that the expression of HMGCS1 in cervical cells is inhibited, and the total cholesterol is reduced, and proliferation, migration and invasion of cervical cancer cells are inhibited.
The invention also provides application of the reagent for inhibiting the expression of Rad17 in preparing a medicament for treating cervical cancer.
In the above application technical scheme, the agent for inhibiting the expression of Rad17 inhibits the expression of Rad17, thereby inhibiting the expression of HMGCS1 in cervical cells, thereby reducing total cholesterol and inhibiting proliferation, migration and invasion of cervical cancer cells.
In the above application technical scheme, the agent for inhibiting Rad17 expression includes a nucleic acid molecule, a protein molecule or a compound; preferably shRNA.
The beneficial effects of the invention are as follows: the novel cervical cancer diagnosis marker Rad17 is provided, the occurrence and the development of cervical cancer can be clearly and clearly represented, and the marker presents a specific high-expression phenomenon in clinical cervical cancer tissues of human compared with the corresponding normal cervical tissues; high expression is also exhibited in human cervical cancer cell lines; and it was demonstrated that knocking down Rad17 suppresses cervical cancer malignancy. Rad17 is used as a diagnosis marker of cervical cancer, provides a new molecular target for the treatment of cervical cancer, and provides a new idea for the treatment of cervical cancer.
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FIG. 1 is an analysis of the expression of Rad17 in human normal cervical tissue and human cervical cancer tissue in GEO database.
FIG. 2 is an analysis of the effect of Rad17 on disease-free survival of cervical cancer patients using a GEPIA online database.
FIG. 3 is an RT-qPCR assay for the mRNA levels of Rad17 in human normal cervical tissue and human cervical cancer tissue.
FIG. 4 shows IHC detection of protein expression levels of Rad17 in human normal cervical cancer tissue, CIN tissue, and cancer tissue.
FIG. 5 is a graph showing the western blot detection of protein expression levels of Rad17 in immortalized human normal cervical epithelial cells (H8) and several human cervical cancer cell lines (CaSki, siHa and HeLa).
FIG. 6 shows the relative amounts of mRNA expressed by RT-qPCR for Rad17 in immortalized human normal cervical epithelial cells (H8) and several human cervical cancer cell lines (CaSki, siHa and HeLa).
FIG. 7 is a graph of the knockdown efficiency of Rad17 in CaSki and SiHa verified by western blot and RT-qPCR.
FIG. 8 is a graph of RTCA testing proliferation potency after knock-down of Rad17 in CaSki and SiHa.
FIG. 9 is a graph of the proliferation potency of CCK8 in detecting the knockdown of Rad17 in CaSki and SiHa.
FIG. 10 is a cloning experiment to examine proliferation capacity after knocking down Rad17 in CaSki and SiHa.
FIG. 11 is a scratch test to examine the migration ability of CaSki and SiHa after knocking down Rad17.
FIG. 12 is a Transwell experiment to examine migration and invasion capacity after Rad17 knockdown in CaSki and SiHa.
Fig. 13 is a GEPIA database analysis of correlation of HMGCS1 and Rad17 in cervical cancer tissue.
FIG. 14 shows protein and mRNA levels of HMGCS1 after knock-down of Rad17 in CaSki by western blot and RT-qPCR.
FIG. 15 is a graph showing total cholesterol assay to determine total cholesterol levels in CaSki after Rad17 knockdown.
FIG. 16 is a graph showing total cholesterol levels in CaSki after over-expressing Rad17 and after treatment of the over-expressing Rad17 cells with the HMGCS1 inhibitor Dipyridamole.
Fig. 17 is a GEO database analysis of HMGCS1 expression in cervical cancer.
FIG. 18 is a graph showing the protein and mRNA expression levels of HMGCS1 in immortalized human normal cervical epithelial cells and several human cervical cancer cell lines by western blot and RT-qPCR.
FIG. 19 is a western blot to detect knockdown effects of HMGCS 1.
Fig. 20 is a total cholesterol quantification assay to detect total cholesterol content after HMGCS1 knockdown in CaSki.
FIG. 21 is a graph showing the proliferation potency of CCK8 after knocking down HMGCS1 in CaSki and SiHa.
FIG. 22 is a Transwell experiment to examine migration and invasion capacity after knock-down of HMGCS1 in CaSki and SiHa.
Fig. 23 is a comparison of tumor tissue volume down-regulating Rad17 versus control tumor tissue volume.
FIG. 24 is a comparison of tumor tissue weight down-regulating Rad17 versus control tumor tissue weight.
Fig. 25 is the change in HMGCS1 at day 15 of IHC assay for engraftment.
Detailed Description
The invention is further illustrated, but is not limited, by the following examples.
The experimental methods in the following examples are conventional methods unless otherwise specified.
Example 1
1 Experimental materials and methods
1.1 cell culture
The experiment used 3 human cervical cancer cell lines CaSki, siHa and HeLa cells and 1 immortalized normal human cervical epithelial cell line H8.CaSki cell line (Procell, wuhan) was cultured in RPMI-1640 medium (C11875500 BT, gibco) containing 10% fetal bovine serum (C8010, adamas, USA); siHa cells (Procell, wuhan) in the addition of 10% fetal bovine serumIs cultured in MEM-alpha medium (L570 KJ, basalmia); heLa cells (Procell, wuhan) were cultured in DMEM medium (C11995500 BT, gibco) containing 10% fetal bovine serum; h8 cells (green flag, shanghai) were cultured in DMEM medium supplemented with 10% fetal bovine serum. All these cell lines were at 37℃and 5% CO 2 Is cultured in a standard environment.
1.2 lentiviral packaging and construction of cells knocked down Rad17 or HMGCS1
In order to obtain cells with reduced Rad17 expression, shRNA virus containing target Rad17 was prepared by lentiviral packaging as follows: lentiviruses were packaged using a lentivirus packaging three plasmid system, lentivirus packaging plasmid pMD2G, psPAX, and shRad17 (Sigma-Aldrich, USA) or plKO.1-TRC vector (shRad 17 control (NC) plasmid, sigma-Aldrich, USA) at a mass of 2:9:9, and when HEK 293T cells were 70% fused, they were transformed into cells by PEI (24765-1, polysciences) according to the plasmid System product instructions. After 48h, the virus supernatant was collected. When the cells reached 50% confluence, shRNAs virus containing Rad17 as target was transfected into CaSki and SiHa cells, and infection was mediated by polybrene reagent (C0351, beyotime) according to the product instructions. After 48h puromycin (ST 551, beyotime) was used for screening. Cell construction knockdown of HMGCS1 as above, shHMGCS1 sequences are shown in table 1.
TABLE 1shRNA sequences
1.3CCK-8 experiment
The cells were grown at 3X 10 3 The density of individual cells/well was seeded into 96-well plates, and 5 duplicate wells were placed in each group. Subsequently, 10. Mu.L of CCK-8 solution was added to each well at 0, 24, 48, 72 and 96h, respectively, and incubated at 37℃for 1.5h. Absorbance at a wavelength of 450nm was measured using a microplate reader. Cell viability was plotted from absorbance values.
1.4 cloning experiments
The cells were grown at 3X 10 3 Density of wells/density of wells was inoculated into six well plates and cells were cultured14 days. Next, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cell phone photographs and counts using Image J software to calculate the clone formation rate.
1.5 immunohistochemical staining (IHC)
Paraffin sections were IHC stained. Paraffin sections were dewaxed in xylene and a series of ethanol of different concentrations. After antigen retrieval, blocking endogenous peroxidase, blocking, and binding to Rad17 antibody (# 13358-1-AP, 1:200 Proteintech, USA) was carried out overnight at 4 ℃. The enzyme-labeled goat anti-mouse/rabbit IgG polymer secondary antibody (PV-6000, zsbio, china) was incubated for 20min at room temperature. Then, DAB color development, hematoxylin staining, graded ethanol dehydration and neutral resin sealing. 1.6 Real Time Cell Analysis (RTCA)
For RTCA, cells were at 3X 10 3 Cell/well density was seeded in E-Plate Insert 16 (6465382001, agilent, USA) and real-time cell index was measured by xCELLigence RTCA S.
1.7 real time quantitative polymerase chain reaction (RT-qPCR).
Total RNA in cervical cancer cell lines was extracted using RNAiso Plus (9109, takara, beijing) and reverse transcribed into cDNA templates using ABScript III RTMaster Mix for qPCRwith gDNARemover (RK 20429, ABclonal, wuhan). Gene ID 5884 in NCBI of Gene Rad17, gene ID 3157 in NCBI of Gene HMGCS1, and designed specific primer sequences (shown in Table 2) were synthesized by Tsingke corporation (China). Subsequently, cDNA templates, specific primers, SYBR Green Fast qPCR Mix reagents (RK 21203, ABclonal, china) were mixed to amplify the target gene, and RT-qPCR reactions were performed in Roche LightCycler II system. Rad17 or HMGCS1 expression was normalized to GAPDH or β -actin. The relative expression was calculated using the 2- ΔΔCT method.
TABLE 2RT-qPCR primer sequences
Using cDNA obtained by reverse transcription as a template, 3 wells were set for each set of samples, and a reaction system was prepared on ice according to the following ingredients.
qPCR reactions were performed as follows:
calculation results: and calculating the mRNA relative expression quantity of the target gene according to the Ct value of the target gene and the Ct value of the reference gene obtained by qPCR reaction and the 2-delta Ct.
1.8 scratch test
Scratch tests were used to assess cell migration capacity. Cells were treated in serum-free medium for 12 hours. Scratches were formed by a 10. Mu.l pipette tip and recorded by an inverted fluorescence microscope (DMi 8, leka, germany). After 24 hours, the cell migration distance was measured using Image J software, and the cell migration capacity was calculated.
1.9Transwell experiments
Cells were assessed for their ability to migrate and invade using a Transwell method. For migration experiments, cell suspensions (6×10 4 Individual cells/wells) were seeded in the upper chamber of a Transwell (14341, labselect, anhui). For invasive experiments, cell suspensions (7.5X10 4 Individual cells/well) was inoculated into a Transwell upper chamber, and matrigel (356234,BD Biosciences,San Jose, usa) was diluted 1:9 with serum-free medium, pre-coated 1h in advance to the upper chamber. 600ul of medium containing 20% fetal bovine serum was added to the lower chamber, and after 48 hours incubation, the cells in the upper chamber were taken and washed 2 times with PBS. Cells were then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (G1014, servicebio, maroon). The number of cells in the lower chamber was observed using a positive microscope, and 3 fields were randomly selected from each image for counting.
1.10 quantitative determination of Total cholesterol
The quantitative measurement of total cholesterol in cells was carried out according to the instructions of the total cholesterol (T-CHO) test box (A111-1-1, built in Nanjing, china).
1.11 immunoblotting
Total protein of cervical cancer cell lines was isolated using PMSF-containing RIPA lysate (R0010, solaro, china) and quantified using BCA assay kit (P0010, beyotime, china). Equal amounts of the proteins were then loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto PVDF membranes (ISEQ 00010, millipore, china). After blocking for 1h at room temperature with 5% skim milk, the membrane was incubated overnight at 4℃with one of the following specific antibodies: anti-Rad17 anti-body (1:6,000,13358-1-AP; proteintech, USA), GAPDH (1:10000; 60004-1-Ig, proteintech). Subsequently, HRP bound goat anti-rabbit antibody (1:10000; 5220-0336, seracare, USA) or HRP-labeled goat anti-mouse antibody (1:10000; 5220-0341, seracare) was incubated with the membrane for 1h at room temperature. Protein images were collected using ECL kit (AP 34L024/AP34L025, life-ilab, china) and integrated imager (ImageQuant LAS 500,GE Healthcare Life Sciences), quantified using Image J software, using β -actin or GAPDH as internal reference.
1.12 xenograft tumor model analysis
This study was approved by the ethics committee of Chongqing medical university. All animal experiments were performed according to the guidelines for laboratory animal care and use. Balb/c nude mice (4-5 weeks, 18-20 g) were purchased from Hunan Style Levoda laboratory animals Co., ltd., china and placed in a standard environment. sh-Rad17 or sh-NC (control) cells were stably transfected using puro antibiotics (ST 551, beyotime). Then, 7X 10 per animal 6 Density of individual cells sh-Rad17 or sh-NC stably transfected SiHa cells were subcutaneously injected to the left of the back. According to tumor volume (mm) 3 ) = (length x width 2 ) The formula of/2 measures tumor volume with calipers every 3-5 days. Mice were sacrificed 15 days later and tumor weights were measured.
1.13 statistical analysis
Data are expressed as mean ± standard deviation, analyzed using GraphPad Prism software (san diego, california). P <0.05 using t-test or one-way anova comparison after Bonferroni test.
2 results
Abnormal upregulation of expression of 1R ad17 in cervical cancer
We first examined the expression of Rad17 in cervical cancer and its effect on the disease-free survival of cervical cancer patients. We analyzed the expression of Rad17 in 14 normal cervical tissues and 14 HPV 16-positive cervical cancer tissues in the GEO database, as shown in FIG. 1, the expression of Rad17 in HPV 16-positive cervical cancer tissues was significantly higher than in normal tissues. GPIA database survival analysis showed (FIG. 2) that Rad17 high expressing cervical cancer patients had shorter disease-free survival than low expressing patients. This suggests that our Rad17 may affect the development of cervical cancer. Furthermore, we detected Rad17mRNA levels in 11 normal cervical tissues and 18 cancer tissues by quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis. FIG. 3 shows that Rad17 expression in cervical cancer tissue is significantly increased. To verify the expression of Rad17 at the protein level, an additional 15 human cervical tissue samples were subjected to Immunohistochemical (IHC) analysis, including 5 normal, 5 precancerous lesions and 5 cervical cancer tissue samples, using Rad17 specific antibodies. The results showed that the staining intensity of Rad17 was much higher in cancer tissues than in normal tissues, indicating that Rad17 was significantly up-regulated in cervical cancer tissues (fig. 4). Rad17 expression was then detected by RT-qPCR and western blot in several cervical cancer cell lines (such as CaSki, siHa and HeLa). Cervical cancer cells exhibited higher Rad17mRNA and protein levels (fig. 5 and 6) than immortalized normal cervical epithelial cell line (H8), consistent with our observations at the tissue level.
2.2 knockdown of Rad17 inhibits proliferation, migration and invasion of cervical cancer cells
To examine whether Rad17 regulates the development of cervical cancer, shRNA knockdown Rad17 was used to examine the effect on cell proliferation. Knockdown cells were generated from CaSki or SiHa cell lines transduced with two different lentiviruses expressing Rad17-shRNA and stably screened by puromycin. The western blot or RT-qPCR analysis in FIG. 7 shows that these generated cells have high knockdown efficiency. The effect of Rad17 on cell growth was determined using real-time cell analysis (RTCA). Our results indicate that the growth rate of the knockdown group is significantly lower than that of the control group (fig. 8). Similarly, CCK-8 analysis showed that Rad17 silencing significantly inhibited cell proliferation of cervical cancer cells (fig. 9). Likewise, the Rad17 knockdown group had fewer colonies than the control group (fig. 10).
In addition to cell proliferation, we tested the role of Rad17 in cervical cancer cell migration and invasion. The knockdown of Rad17 was shown to greatly reduce the rate of wound healing by comparing the knockdown cells with control cells using a scratch healing experiment (fig. 11).
Similarly, transwell migration and invasion experiments were used to examine the effect of knockdown Rad17 on cell migration and invasiveness. Fig. 12 shows that fewer cells migrated in the Rad17 knockout group compared to the control group. Furthermore, after knocking down Rad17, the number of invading cells was significantly reduced (fig. 12). Taken together, these results indicate that Rad17 promotes proliferation, migration and invasion of cervical cancer cells.
2.3 knockdown of Rad17 inhibits expression of HMGCS1 in cervical cancer cells
As shown in fig. 13, GEPIA database analysis showed that HMGCS1 is highly correlated with Rad17 in cervical cancer tissues, so we hypothesize that Rad17 affects the occurrence and development of cervical cancer by modulating HMGCS 1. RT-qPCR and western blot experiments demonstrated that knocking down Rad17 reduced the mRNA and protein levels of HMGCS1 in CaSki cells, FIG. 14. Since HMGCS1 is one of the key enzymes in the cholesterol synthesis pathway, HMGCS1 catalyzes the conversion of acetoacetyl-CoA to HMG-CoA, we examined whether Rad17 regulates cholesterol biosynthesis by modulating HMGCS 1. As shown in fig. 15, silencing Rad17 down-regulates total cholesterol levels in CaSki cells. Furthermore, the HMGCS1 indirect inhibitor Dipyridamole partially counteracts the Rad 17-induced increase in total cholesterol content, fig. 16. Taken together, these data indicate that Rad17 affects the development of cervical cancer by modulating 3-hydroxy-3-methylglutaryl coa synthase 1 (HMGCS 1).
2.4 silencing HMGCS1 reduces Total cholesterol and inhibits proliferation and migration of cervical cancer cells
The link between HMGCS1 and cholesterol metabolism is widely accepted. However, it is not clear whether HMGCS1 would cause cervical cancer. Recent studies indicate that HMGCS1 is closely related to cancer. Therefore, we intend to investigate whether HMGCS1 would affect cervical cancer. The GEO database shows high expression of HMGCS1 in cervical cancer tissue, fig. 17.Western blot and RT-qPCR showed that the expression levels of HMGCS1 protein and mRNA in cervical cancer cells (CaSki, siHa and HeLa) were significantly higher than in normal cervical epithelial cells (H8), FIG. 18. We produced stable HMGCS1 knockdown cervical cancer cells. Western blot analysis confirmed the lentiviral infection efficiency of HMGCS1, FIG. 19. We first tested the effect of HMGCS1 on cholesterol metabolism. The results showed that the knock-down HMGCS1 reduced the total cholesterol content of CaSki cells, figure 20.
To verify whether HMGCS1 affects cervical cancer growth, CCK8 experiments were performed, which indicated that silencing HMGCS1 significantly inhibited cervical cell growth, fig. 21.Transwell migration and invasion experiments showed that knockdown HMGCS1 inhibited the migration and invasion capacity of cervical cancer cells, fig. 22.
2.5 knockdown of Rad17 inhibits the formation of xenograft tumors
To test the effect of Rad17 on tumor growth in vivo, siHa cells were stably transfected with shRad17 and subcutaneously injected into the left dorsal portion of nude mice. As shown in fig. 23 and 24, the size and weight of the tumor can be suppressed by down-regulating Rad17. As shown in fig. 25, knocking down Rad17, expression of HMGCS1 protein level was significantly inhibited. In summary, rad17 also affects cholesterol synthesis in vitro by affecting HMGCS1, thereby affecting the growth of cervical cancer cell transplants.
The research of the invention shows that: 1) Rad17 is expressed in cervical cancer tissues to increase and is associated with poor prognosis. 2) Specific knockdown of Rad17 inhibits malignant progression of cervical cancer in vitro and in vivo. 3) Rad17 regulates cholesterol synthesis by regulating HMGCS1 expression and thus affects the malignant progression of cervical cancer.
Claims (10)
1. Use of a substance for detecting the expression level of a biomarker for the manufacture of a product for diagnosing cervical cancer, characterized in that: the biomarker is Rad17.
2. The use according to claim 1, characterized in that: detecting the level of Rad17 gene or protein expression in cervical tissue of an individual suffering from or suspected of suffering from cervical cancer.
3. The use according to claim 1, characterized in that: if the level of Rad17 gene or protein expression in diseased cervical tissue of an individual exhibits a specific high expression compared to normal cervical tissue, it is indicative that the individual is a high risk patient for cervical cancer.
4. The use according to claim 1, characterized in that: the product is a reagent or a kit.
5. The use according to claim 1, characterized in that: the substance is used for detecting the protein expression level of the Rad17 gene or the mRNA expression level of the Rad17 gene.
The application of rad17 as a target in screening/preparing a medicament for treating cervical cancer is characterized in that: inhibiting the expression of Rad17 gene and inhibiting the malignant characteristics of cervical cancer.
7. The use according to claim 6, characterized in that: inhibiting the expression of Rad17, thereby inhibiting the expression of HMGCS1 in cervical cells, thereby reducing total cholesterol and inhibiting proliferation, migration and invasion of cervical cancer cells.
8. Application of an agent for inhibiting Rad17 expression in preparing a medicament for treating cervical cancer.
9. The use according to claim 8, characterized in that: agents that inhibit Rad17 expression, thereby inhibiting HMGCS1 expression in cervical cells, thereby reducing total cholesterol and inhibiting proliferation, migration, and invasion of cervical cancer cells.
10. The use according to claim 8, characterized in that: the agent for inhibiting Rad17 expression comprises a nucleic acid molecule, a protein molecule or a compound; preferably shRNA.
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| CN202310305905.9A CN116356028A (en) | 2023-03-27 | 2023-03-27 | Application of Rad17 as cervical cancer marker and therapeutic target |
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| CN202310305905.9A CN116356028A (en) | 2023-03-27 | 2023-03-27 | Application of Rad17 as cervical cancer marker and therapeutic target |
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