CN106544421B

CN106544421B - Application of SPAG6 gene as ovarian tumor diagnosis and treatment marker

Info

Publication number: CN106544421B
Application number: CN201610921519.2A
Authority: CN
Inventors: 张志兵; 石玉琴; 张玲; 柳赟昊; 付国庆; 江高峰; 周婷; 李玉红
Original assignee: Wuhan University of Science and Engineering WUSE
Current assignee: Wuhan University of Science and Engineering WUSE
Priority date: 2016-10-21
Filing date: 2016-10-21
Publication date: 2020-05-08
Anticipated expiration: 2036-10-21
Also published as: CN106544421A

Abstract

The invention relates to an application of SPAG6 gene in diagnosis and treatment of ovarian tumor. The invention proves that the SPAG6 gene has differential expression in ovarian tumor tissues and normal tissues by using QPCR and Western blot methods, detects the influence of the SPAG6 gene on the reproductive development of ovarian cancer cells SKOV-3 by using an in vitro growth curve and a paclitaxel growth inhibition test, and can be used as an index for early diagnosis of ovarian tumors by using SPAG 6. Through the experiment of SPAG6 on ovarian epidermal tumor, the invention provides a new target for diagnosis and treatment of ovarian tumor and the like, and is used for guiding the research and development of new drugs so as to improve the cure rate and survival rate of tumor patients.

Description

Application of SPAG6 gene as ovarian tumor diagnosis and treatment marker

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of SPAG6 gene in diagnosis and treatment of ovarian tumor.

Background

Microtubules are a ubiquitous fibrous structure of eukaryotic cells, formed by the polymerization of tubulin and small amounts of microtubule-associated proteins, and are one of the major components of the cytoskeleton. Microtubules play an important role in vivo, and are mainly involved in cell movement, organelle localization, intracellular material transport, eukaryotic mitosis process, cell signaling, and the like.

Intracellular microtubules exist as both cytoplasmic microtubules and spindle microtubules, which are dynamically balanced between dynamically assembled and disassembled states in different cell cycles, and any factor that imbalances this balance can disrupt the microtubule structure, thereby affecting cell function. Tumor cells have the ability to proliferate rapidly, with frequent mitotic processes and significantly shorter cell cycles than normal cells. Therefore, if the dynamic cycle is destroyed, the mitosis process of the tumor cell is influenced, the growth of the tumor cell is inhibited or the apoptosis of the tumor cell is induced, so that the microtubule is the best target point for tumor treatment. The effects of drugs on microtubules can be divided into two categories, depending on the mechanism by which the tubulin/microtubule cycle is disrupted: promote tubulin polymerization and stabilize the formed microtubules (inhibit microtubule depolymerization) and inhibit tubulin polymerization. The former can aggregate tubulin to stabilize the tubulin complex, and the latter inhibits tubulin polymerization to dissociate microtubules. The binding cavity of the microtubule stabilizing drug on tubulin is close to the important action region between heterodimer and protofilament, thus the drug is combined with tubulin and simultaneously the combination between heterodimer and protofilament is strengthened, consequently, the polymerization of tubulin is promoted, and formed microtubules can be stabilized, and the cell division is stopped at C2/M stage. Examples of such drugs include taxol. The latter may inhibit polymerization of tubulin into microtubules, or depolymerize already polymerized microtubules.

At present, microtubule-associated drugs such as paclitaxel have played an important role in the treatment of ovarian cancer tumors, but drug tolerance and side effects such as anaphylaxis, bone marrow suppression, cardiovascular and cerebrovascular system lesions, nausea, vomiting, arthralgia, alopecia and the like limit the drugs to play a greater role. Therefore, it is important to explore new formulations and to find new microtubule-binding target proteins.

The early stage lesion of the ovarian tumor is atypical, so that the condition of the ovarian tumor is easily overlooked or delayed due to improper treatment, and the ovarian tumor is usually diagnosed in the later stage clinically. Therefore, the search for molecular biomarkers suitable for early diagnosis and evaluation of tumor prognosis of malignant tumors becomes a hot point of research.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention aims to provide a molecular marker for early diagnosis of ovarian tumor.

Preferably, it should be noted that the present invention was unexpectedly discovered based on the following studies and investigations.

The current research shows that the sperm-associated antigen (SPAG) is highly expressed in ovarian tumor, the expression of SPAG6 is regulated by the methylation level of a promoter thereof, the SPAG6 gene silencing obviously inhibits the growth of ovarian cancer cells, and the influence on the biological characteristics of the tumor cells after the gene is inhibited is avoided. The research results show that SPAG6 can be a new target protein for ovarian tumor diagnosis and treatment, and therefore has great application value.

In order to research whether the expression of the SPAG6 gene is increased in the ovarian epidermal tumor tissue, 40 ovarian cancer patient samples and 40 cancer adjacent tissue control samples are taken by the inventor, and the expression level of SPAG6 in ovarian cancer is obviously higher than that of the cancer adjacent tissue under a microscope by using an immunohistochemical method. The methylation of the SPAG6 gene promoter is detected by a pyrophosphate sequencing method, the methylation conditions of 76 CpG islands at 13 sites in the SPAG6 promoter region are detected, and 15 abnormally methylated CpG islands are found, wherein 4 of the CpG islands are hypermethylated and 11 of the CpG islands are hypomethylated. The mRNA and protein expression of SPAG6 of normal human ovarian epithelial cells and SKOV-3 cells is detected by using real-time quantitative PCR and western blot, and the result shows that the mRNA and protein expression level of SPAG6 in the SKOV-3 cells is obviously higher than that of the normal human ovarian epithelial cells.

In order to research the influence of the demethylation reagent 5-AZA on the mRNA expression of the SPAG6 gene of the ovarian epithelial cell, the inventor uses 5μm/L demethylation reagent 5-AZA to treat the normal human ovarian epithelial cell for 48 hours, extracts total RNA, and detects the mRNA expression level of the SPAG6 gene by real-time quantitative PCR, and experiments show that the expression of the treated SPAG6 mRNA is obviously increased compared with that of the untreated cell.

In order to research the influence of the low expression of the SPAG6 gene on the malignant biological characters of tumor cells, the inventors designed 4 RNA interference fragments targeting SPAG6 and inserted the RNA interference fragments into a pYr-LVsh vector. After the cells are transfected, total RNA is extracted, and the SPAG6 mRNA expression level is detected by a real-time quantitative PCR method. Through screening, the inventor finds that the inhibition rate of the SPAG6-sh4 sequence is higher and is 91%. We have subsequently constructed RNA-interfering lentiviruses targeting SPAG6, and after infection of cells, the results indicated interference efficiencies between 19% and 91%. The shRNA4 plasmid is transfected into SKOV3 ovarian cancer cells, a SPAG6 low-expression cell strain is established, a cell growth curve is drawn, and the cell growth speed is found to be remarkably reduced.

To demonstrate that SPAG6 gene inhibition increases tumor cell sensitivity to paclitaxel (Taxol), the inventors seeded SPAG6 suppressor cells and control cells in 24-well plates, followed by treatment with Taxol (0, 1, 10, 50 nM). After 3 days, viable cells were counted and survival rate was calculated. The results show that after SPAG6 of SKOV-3 cells is inhibited, the cell survival rate of the group with the dose of more than 10nM is obviously reduced, and the sensitivity of tumor cells to the treatment of Taxol is increased after SPAG6 is inhibited.

Therefore, in the first aspect of the invention, the invention provides the application of the product for detecting the expression of the SPAG6 gene in the preparation of a tool for diagnosing ovarian tumors.

According to the embodiment of the invention, the expression of the SPAG6 gene is different in the normal tissue and the ovarian tumor tissue.

According to the embodiment of the invention, the expression level of the SPAG6 protein in ovarian epidermal cell tumor tissue is as follows: SPAG6 was expressed at a significantly higher level in tumor tissues than in paraneoplastic tissues.

According to the embodiment of the invention, the expression of the SPAG6 gene is different in the normal ovarian epithelial cells and the ovarian cancer epithelial cells.

According to the embodiment of the invention, the differential expression of the SPAG6 gene is detected on the transcription level, and the mRNA expression level of the SPAG6 gene in ovarian cancer epithelial cells is obviously increased compared with normal ovarian epithelial cells.

According to the embodiment of the invention, the differential expression of the SPAG6 protein is detected at the protein level, and the SPAG6 protein content in ovarian cancer epithelial cells is increased compared with normal ovarian epithelial cells.

According to the embodiment of the invention, the influence of the expression inhibition of the SPAG6 gene on the malignant biological traits of the tumor cells comprises the following steps:

the recombinant plasmid GFP-Spag6L/pcDNA3-Spag6L transfects CHO cells/COS-7 cells respectively;

four target sequences were designed from the SPAG6 mRNA sequence: shRNA1, shRNA2, shRNA3 and shRNA4, which were transferred into PLV RNAi plasmids following the instructions;

after transfecting cells, extracting total RNA;

as a result:

the real-time quantitative PCR detection of the SPAG6 mRNA expression level has higher SPAG6-sh4 sequence inhibition rate;

western blot detection shows that the protein expression of the shRNA4-spag6 stable cells is obviously reduced.

According to an embodiment of the invention, the SPAG6 gene suppression increases the sensitivity of tumor cells to taxol.

According to the embodiment of the invention, the expression of the SPAG6 gene in ovarian tumor tissues adopts a pyrophosphate sequencing method, and the method for detecting the methylation of the SPAG6 promoter CpG island by the pyrophosphate sequencing method is as follows:

step 1: carrying out methylation modification;

step 2: purifying sulfite-modified DNA;

and step 3: methylation PCR;

and 4, step 4: pyrosequening assay.

According to the embodiment of the invention, the detection steps of the expression level of the SPAG6 protein in ovarian epidermal cell tumor tissue are as follows:

step 1: paraffin section is dewaxed to water;

step 2: 3% H₂O₂Incubating at room temperature for 5-10min to eliminate the activity of endogenous peroxidase;

and step 3: washing with distilled water, and soaking in PBS for 5min for 2 times;

and 4, step 4: blocking with 5-10% normal goat serum (PBS dilution), incubating at room temperature for 10min, removing serum, and washing; dripping primary antibody working solution, and incubating at 37 ℃ for 1-2 h;

and 5: washing with PBS for 3 times in 5 min;

step 6: dripping appropriate amount of biotin-labeled secondary antibody working solution, and incubating at 37 deg.C for 10-30 min;

and 7: washing with PBS for 3 times in 5 min;

and 8: adding appropriate amount of horseradish enzyme or alkaline phosphatase labeled streptavidin working solution dropwise, incubating at 37 deg.C for 10-30min

And step 9: washing with PBS for 3 times in 5 min;

step 10: developing with color developing agent for 3-15 min;

step 11: fully washing with tap water, re-dyeing, dewatering, transparentizing and sealing.

According to an embodiment of the invention, the method for detecting the differential expression of the SPAG6 gene at the transcriptional level comprises the following steps:

step 1: extracting total RNA;

step 2: reverse transcription;

and step 3: synthesizing a target gene PCR primer;

and 4, step 4: statistical analysis is performed by using SPSS20.0 statistical software;

and 5: and (6) obtaining the result.

According to an embodiment of the invention, the method for detecting the differential expression of SPAG6 at the protein level is as follows: the protein extraction procedure was performed according to the instructions of the EpiQuik tissue/cell total protein extraction kit.

The comprehensive scheme of the invention indicates that the expression of the SPAG6 gene is changed in ovarian tumor, and the methylation degree of a promoter CpG island of the SPAG6 gene has influence on the expression of the SPAG6 gene; the expression of the knock down SPAG6 gene in SKOV-3 cells proves the potential application value of the gene in the treatment of ovarian epidermal tumor. Through the experiment of SPAG6 on ovarian epidermal tumor, the invention provides a new target for diagnosis and treatment of ovarian tumor and the like, and is used for guiding the research and development of new drugs so as to improve the cure rate and survival rate of tumor patients.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 shows the location distribution of abnormal methylated CpG island sites in the SPAG6 promoter region of ovarian cancer tissue.

FIG. 2 shows the sequencing diagram of the CpG island of the promoter region of the SPAG6 gene.

FIG. 3 shows SPAG6 protein expression in ovarian cancer tissue by immunohistochemical method.

FIG. 4 shows SPAG6 gene mRNA expression levels in ovarian cancer cells versus normal cells.

FIG. 5 shows SPAG6 gene protein expression levels in ovarian cancer cells versus normal cells.

FIG. 6 pGreen-Puro CMV plasmid map.

FIG. 7 shows the inhibition of mRNA expression efficiency by targeting the SPAG6 gene shRNA.

FIG. 8 shows that the shRNA4 of the targeted SPAG6 gene inhibits protein expression.

FIG. 9 shows the effect of inhibition of SPAG6 expression on tumor cell growth.

FIG. 10 shows that SPAG6 gene inhibition increases the sensitivity of tumor cells to paclitaxel (Taxol).

Detailed Description

The following describes embodiments of the present invention in detail. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

Example 1 difference in expression of the SPAG6 gene in normal tissue and ovarian tumor tissue.

1. Materials and methods.

1.1 origin of patients.

Tumor and paracancer normal tissue specimens of patients with ovarian cancer surgery are collected by multiple hospitals in Wuhan City. All patients signed informed consent and were approved by the medical ethics committee of the university of wuhan science and technology.

2. The pyrosequencing method detects methylation of CpG islands of the SPAG6 promoter.

2.1 materials.

2.1.1 reagents and kits.

。

2.1.2 apparatus.

。

2.2 methods.

2.2.1 methylation modification

(1) Dissolving DNA with sulfite reagent, adding 800 μ l of RNase-free water into each sulfite mixture, and mixing;

(2) the following reagents were prepared as in Table1 in 200. mu.l PCR thin walled tubes;

table1. sulfite modification reaction component

Composition (I)	Volume (μl)
		DNA solution (1-2ug)	Variable (maximum 40ul)
Rnase-free water	Variable
		Sulfite mixed liquor (dissolved)	85
DNA protective solution	15
		Total volume	140

Total volume of DNA and water must be 40 ul;

(3) covering the tube cover and thoroughly mixing, and standing at room temperature (14-25 ℃);

(4) DNA transformation was carried out using a PCR machine, and the reaction conditions were set as shown in Table 2 (about 5 hours)

Step	Time(min)	Temperature(℃)
			Denaturation of the material	5min	95℃
Renaturation	25min	60℃
			Denaturation of the material	5min	95℃
Renaturation	85min(1h25min)	60℃
			Denaturation of the material	5min	95℃
Renaturation	175min(2h55min)	60℃
			Hold	Indefinite word	20℃

The reaction system can be placed in a PCR instrument overnight

(5) The PCR thin walled tube was placed in a PCR instrument and the cycle was started.

2.2.2 purification of sulfite-modified DNA

(1) After the sulfite modification is finished, centrifuging the PCR thin-walled tube, and transferring the reaction solution into a 1.5ml centrifuge tube;

(2) 560ul of freshly prepared Buffer BL (containing 10ug/ml carrier RNA) was added to each tube, vortexed and briefly centrifuged;

(3) EpiTect spin columns were placed in a collection tube and the solution from the previous step was transferred to the column;

(4) centrifuging at the highest speed for 1min, discarding the filtrate, and placing the column back into the collection tube;

(5) 500ul Buffer BW (wash Buffer) was added to each column, centrifuged at maximum speed for 1min, the filtrate removed and the column returned to the collection tube;

(6) 500ul Buffer BD (desulfonated solution) was added to each column and incubated for 15min at room temperature (15-25 deg.C);

(7) centrifuging at the highest speed for 1min, discarding the filtrate, and placing the column back into the collection tube;

(8) 500ul Buffer BW was added to each column, centrifuged at maximum speed for 1min, the filtrate removed and the column returned to the collection tube;

(9) repeating the step (8) once;

(10) the column was placed in a new 2ml collection tube and centrifuged at maximum speed for 1min to remove all residual liquid;

(11) the lid of the column was opened and placed in a new 1.5ml centrifuge tube and incubated at 56 ℃ for 5 min;

(12) the column was placed in a new 1.5ml centrifuge tube, 20ul buffer EB was added drop wise to the center of the membrane of the column and centrifuged at maximum speed for 1min to elute the purified DNA.

2.2.3 methylation PCR

(1) Primer synthesis was designed by PyroMark Assay Design 2.0, with the primer information shown in table 1:

TABLE1 sulfite sequencing PCR primer information SEQ ID NO

(2) PCR reaction system (50ul)

；

(3) Reaction conditions

。

2.2.4 Pyrosequening assay

(1) Adding 2ul of reaction binding beads, 38ul of binding buffer solution and 40ul of PCR products into a 96-well PCR reaction plate, and fully and uniformly mixing for 10min at room temperature;

(2) starting a vacuum pump to absorb the mixed suspension of the binding beads and the PCR product, and sequentially immersing the mixed suspension into 70% ethanol, 0.2M NaOH and washing buffer solution for 5S respectively;

(3) turning off the vacuum pump, placing the binding beads on the probe and the PCR product in 40ul annealing buffer solution (containing 1.5ul of sequencing primer), denaturing at 85 ℃ for 2min, cooling to room temperature, and annealing and hybridizing the primer and the template;

(4) calculating the dosage according to Pyrosequencing software sequence design information, and sequentially adding a substrate mixture, an enzyme mixture and four dNTPs (QIAGEN) into a reagent cabin;

(5) placing the reagent cabin and the 96-hole reaction plate into a Pyrosequencing detector (PyroMark Q96ID, QIAGEN) for reaction, and automatically analyzing the methylation state of each locus by Pyro Q-CpG software;

sample sequence

Company number original number the company number original number

The sequence of the SPAG6 promoter region of ovarian cancer tissue is SEQ IN NO: 5, through pyrosequencing analysis, the methylation condition of 76 CpG islands at 13 sites in the SPAG6 promoter region is detected, and 15 abnormal methylated CpG islands are found, wherein 4 hypermethylation islands and 11 hypomethylation islands are found; as shown in fig. 1, row 1 and 2 grey represent hypermethylated CpG islands, and row 3 and 4 grey represent hypomethylated CpG islands.

SPAG6 protein expression levels in ovarian epidermal cell tumor tissue.

3.1 specific methods

(1) Paraffin section is dewaxed to water;

(2)3％H₂O₂incubating at room temperature for 5-10min to eliminate the activity of endogenous peroxidase;

(3) washing with distilled water, and soaking in PBS for 5min for 2 times;

(4) blocking with 5-10% normal goat serum (PBS dilution), incubating at room temperature for 10min, removing serum, and washing; dripping primary antibody working solution, and incubating at 37 ℃ for 1-2 h;

(5) washing with PBS for 3 times in 5 min;

(6) dripping appropriate amount of biotin-labeled secondary antibody working solution, and incubating at 37 deg.C for 10-30 min;

(7) washing with PBS for 3 times in 5 min;

(8) dripping appropriate amount of streptavidin working solution labeled by horseradish enzyme or alkaline phosphatase, and incubating at 37 deg.C for 10-30 min;

(9) washing with PBS for 3 times in 5 min;

(10) developing with color developing agent for 3-15 min;

(11) fully washing with tap water, re-dyeing, dewatering, transparentizing and sealing.

3.2 results

Under the observation of a microscope, the expression level of SPAG6 in tumor tissues is obviously higher than that in paracarcinoma tissues in a part of ovarian cancer specimens, and the observation is shown in figure 3.

Example 2 difference in expression of the SPAG6 gene in normal ovarian epithelial cells and ovarian cancer epithelial cells.

1. The differential expression of the SPAG6 gene was detected at the transcriptional level.

1.1 Total RNA extraction

(1) Preparation work: trizol, chloroform (trichloromethane), isopropanol, 75% ethanol containing DEPC (1 thousandth), PBS buffer solution, 1.5ml of EP tube and 200ul of double distilled water containing 1 thousandth DEPC are treated and then dried under high pressure, 1ml of gun head, 200ul of gun head and 10ul of gun head are treated by DEPC water and then dried under high pressure, the DEPC water is prepared according to the proportion of 1:1000 and then is placed for more than 4 hours;

(2) the method comprises the following operation steps: the culture solution was discarded, and 1ml of TRIzol was added, followed by homogenization treatment with a homogenizer. Sample volume should not exceed TRIzol volume 10%. The homogenate was allowed to stand at room temperature (15-30 ℃) for 5 minutes to completely separate the nucleic acid-protein complex. After 5 minutes, 0.2ml of chloroform was added to 1ml of TRIzol, followed by vigorous shaking for 15 seconds, standing at room temperature for 5 minutes, and centrifugation at 4 ℃ for 14000r/min (centrifugation radius of 8.2cm) for 15 minutes, whereby delamination was observed. Carefully taking about 500. mu.l of the upper aqueous phase into another 1.5ml EP tube by using a 200. mu.l tip head, adding 0.5ml isopropanol, mixing well, standing at-20 ℃ for 2h, and centrifuging at 4 ℃ at 14000r/min for 10min to obtain RNA white flocculent precipitate. Removing supernatant, adding 1ml of 75% ethanol containing DEPC, pipetting, mixing, and centrifuging at 4 deg.C at 7500r/min for 10 min. The supernatant was decanted, allowed to stand for several minutes to allow the ethanol to evaporate clean in dry air, 20 μ l of DEPC treated sterile water was added to dissolve the precipitate, and the OD or a value was measured a little and the remainder was stored at-80 ℃.

1.2 reverse transcription

Reverse transcription was performed using the reveral synthesis kit from Fermentas, which had the following composition:

RevertAId H Minus Reverse Transcriptase, RiboLock RNase Inhibitor,5XReaction Buffer, dNTP Mix, oligo (dT)18Primer, Random Hexamer Primer, Control RNA, Control Primers and nucleic-free water;

mu.l of oligo (dT)18 and 4. mu.l of RNA sample (ensuring 2-5. mu.g total RNA) were taken and supplemented with nuclease-free high-purity water to a total volume of 24. mu.l. After mixing well on a shaker, incubation for 5min at 65 ℃ on a PCR instrument, then adding 4. mu.l of 10 Xmm dNTP mix, 8. mu.l of 5Xreaction Buffer, 2ul of RNase inhibitor (RiboLock), 2. mu.l of M-MuLV recombinant Reverse Transcriptase RevertAID H Minus Reverse Transcriptase Transcriptase 2. mu.l on ice, shaking and centrifuging, Reverse transcription was performed on a PCR instrument with the program set as: step 1: 42 ℃ for 60min, step 2: 72 ℃ for 5min, 4 ℃ hold. Finally, 40 mu lcDNA is obtained and is stored at the temperature of minus 20 ℃ after being subpackaged.

1.3 primer design and synthesis of target gene PCR, the primer sequence is shown in Table 2:

TABLE 2 real-time quantitative PCR primer sequences

。

The real-time quantitative PCR (real time PCR) detection is carried out on ABI 384-well fluorescent quantitative PCR plates by adding corresponding reaction system reagents of qPCR mixture (5 mul), ROX reference dye (ROX reference dye)0.2 mul, upstream and downstream primers 0.2 mul respectively, DEPC treated water 2.4 mul, cDNA2 mul, total volume 10 mul, adopting 7900 full-quantitative PCR instrument, using β -actin as internal reference substance, reaction conditions of pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 45s, amplification for 40 cycles, and carrying out relative quantification by delta CT method.

1.4 statistical methods

The experiments are completed according to 3 times of repetition, the result data are all expressed in a mode of mean value plus or minus standard deviation, statistical analysis is carried out by SPSS20.0 statistical software, the difference between different groups is tested by t, and the difference is statistically significant when P is less than 0.05.

1.5 results

As shown in FIG. 4, the mRNA expression level of SPAG6 gene was significantly increased in ovarian cancer epithelial cells as compared with normal ovarian epithelial cells.

2. Differential expression of the SPAG6 gene was detected at the protein level.

2.1. Extraction of Total protein

Carrying out protein extraction operation according to the instruction of an EpiQuik tissue/cell total protein extraction kit; the kit comprises the following components: pre-lysine Buffer; lysine Buffer; balance Buffer; DTT Solution;

2.1.1 Western blot detection

Carrying out SDS-PAGE electrophoresis on the extracted protein quantitatively, and then carrying out membrane transfer, sealing, primary antibody incubation, secondary antibody incubation and color development;

2.1.2 statistical treatment

The grey value of the protein band is analyzed by using Image J software, β -actin is used as an internal reference, the grey value of a target white band is normalized, the result data are expressed in a mode of mean value plus or minus standard deviation, statistical analysis is carried out by SPSS20.0 statistical software, the difference between the two is tested by t, and the difference is considered to have statistical significance when P is less than 0.05;

2.1.3 results

The results are shown in fig. 5, and compared with the normal ovarian epithelial cells, the content of SPAG6 protein in the ovarian cancer epithelial cells is increased, and the difference is statistically significant (P < 0.05).

Example 3 the effect of suppression of SPAG6 gene expression on the malignant biological properties of tumor cells was investigated.

The recombinant plasmid GFP-Spag6L/pcDNA3-Spag6L transfects CHO/COS-7 cells respectively as follows:

(1) the CHO cells/COS-7 cells are inoculated into a six-well plate (six-well plate for immunofluorescence experiment needs to be put into a cell slide in each well in advance) 24 hours in advance until the cell growth density is 50% -70% (1 × 10)⁵One per ml);

(2) two hours before transfection, cells were replaced with fresh cell culture fluid;

(3) according to the concentration of plasmid DNA, 0.8 μ g of plasmid DNA (about 8-10 μ l) is diluted with 25 μ l of DMEM culture solution, and mixed uniformly to prepare a diluent A;

(4) diluting 2. mu.l of transfection reagent with 25. mu.l of cell culture solution, mixing uniformly to prepare a diluent B, and standing at room temperature for 5 minutes;

(5) adding the diluent B into the diluent A completely, mixing uniformly, blowing and sucking up and down for more than 10 times by using a pipettor, and standing at room temperature for 15 minutes;

(6) and (3) adding the diluent into each hole of the culture plate respectively, and shaking uniformly clockwise. Carbon dioxide incubation was performed for 24 hours at constant temperature.

Four target sequences were designed from the SPAG6 mRNA sequence: shRNA1, shRNA2, shRNA3 and shRNA4, which were transferred into PLV RNAi plasmids following the instructions; the SPAG6 mRNA sequence is SEQ IN NO: 9; the target sequences are shown in Table 3:

TABLE 3 shRNA1, shRNA2, shRNA3 and shRNA4 target sequences

。

After transfection of cells, total RNA was extracted, as above, and Spag6 mRNA expression levels were then detected by real-time quantitative PCR. The results showed that the efficiency of interference with expression of Spag6 mRNA was between 19% and 91%. Through screening, the SPAG6-sh4 sequence has higher inhibition rate, as shown in FIG. 7.

Western blot detection results show that: protein expression was significantly reduced in shRNA4-spag6 stable cells, as shown in FIG. 8.

The shRNA4 plasmid is transfected into SKOV3 ovarian cancer cells, a SPAG6 low-expression cell strain is established, a cell growth curve is drawn, and the cell growth speed is found to be remarkably reduced, as shown in figure 9.

Example 4 SPAG6 gene inhibition increases tumor cell sensitivity to taxol.

We seeded SPAG6-shRNA4 stable cells and control cells in 24-well plates and then treated with taxol (0, 1, 10, 50 nM). After 3 days, viable cells were counted and survival rate was calculated. As a result, after SPAG6 inhibition by SKOV-3 cells, the cell survival rate of the group with the dose of more than 10nM was significantly reduced, indicating that the sensitivity to taxol was increased, as shown in FIG. 10.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. The application of the product for detecting the expression of the SPAG6 gene in the preparation of a tool for diagnosing ovarian tumor.