CN109371130B

CN109371130B - Application of RIPOR3 in preparation of biological products for breast cancer detection and treatment

Info

Publication number: CN109371130B
Application number: CN201811373112.6A
Authority: CN
Inventors: 何劲松; 韦伟; 杨子健; 李锋; 刘宝儿; 高睿; 阳莉萍
Original assignee: Peking University Shenzhen Hospital (peking University Shenzhen Clinic Medical College)
Current assignee: Peking University Shenzhen Hospital (peking University Shenzhen Clinic Medical College)
Priority date: 2018-11-19
Filing date: 2018-11-19
Publication date: 2021-08-13
Anticipated expiration: 2038-11-19
Also published as: CN109371130A

Abstract

The invention discloses application of a breast cancer molecular marker RIPOR3 in preparation of a biological product for detection and treatment of breast cancer, and the inventor finds that the expression of RIPOR3 in breast cancer tissues or breast cancer cells is down-regulated. The invention also discloses a kit for breast cancer detection or prognosis, which comprises a primer sequence for specifically amplifying the RIPOR 3. The kit provided by the invention is used for auxiliary diagnosis and prognosis evaluation of breast cancer, and has guiding significance for subsequent clinical treatment. In addition, the application of RIPOR3 in preparing a pharmaceutical composition for treating breast cancer can also be used for clinical application such as gene therapy, drug therapy and the like of breast cancer.

Description

Application of RIPOR3 in preparation of biological products for breast cancer detection and treatment

Technical Field

The invention relates to the technical field of tumor molecular biology, in particular to application of RIPOR3 in preparation of biological products for breast cancer detection and treatment.

Background

Breast cancer has become the most common tumor in women worldwide, and seriously affects the life health of women. Despite the success of the development of screening protocols and adjuvant therapies, a significant proportion of breast cancer patients die from cancer metastasis. Like most other countries, breast cancer has also become the most common cancer among women in china; the new number of Chinese breast cancer accounts for 12.2% of the whole world and the number of deaths accounts for 9.6% of the whole world. Since the 90 s, the incidence rate of breast cancer in China has increased twice more than that of the whole world, and the urban area is particularly remarkable. The key to the diagnosis and treatment of breast cancer lies in early discovery, early diagnosis and early treatment. The activation of oncogenes, or the inactivation of oncogenes, plays a key role in the development of tumors. With the popularization and application of large-scale and high-throughput sequencing technology, the discovery of new oncogenes and cancer suppressor genes not only provides valuable standards for the diagnosis and prognosis of tumors, but also lays a foundation for searching new biological treatment targets of tumors.

The FAM65 protein family includes the members FAM65A (also known as Rho family interacting cell polarization regulator 1), FAM65B (also known as Rho family interacting cell polarization regulator 2) and FAM65C (also known as Rho family interacting cell polarization regulator 3, Rho family-interacting cell polarization regulator 3, RIPOR 3). FAM65A has been shown to localize to podocyte major processes and to the renal cell body. FAM65B is down-regulated early during muscle cell differentiation and occurs in two long and short isoforms that are differentially expressed in different tissues. FAM65C (RIPOR3) appears to occur also in both isoforms, produced by alternative splicing.

The RIPOR3 gene is located at NC _000020.11 of chromosome 20, and the reference to RIPOR3 includes ncbi (genbank) reference sequence transcript variant 1(NM _080829.3) and its encoded shorter protein isoform 1(NP _ 543019.2); and ncbi (genbank) reference sequence transcript variant 2(NM _001290268.1) and its encoded longer protein isoform with a longer N-terminus (NP _001277197.1), variant 2 being different in the 5' UTR and using an alternative upstream AUG compared to variant 1.

At present, the action mechanism of RIPOR3 gene or protein in breast cancer is not clear, and the clinical significance of the function and expression change is not reported. Therefore, the analysis of the relationship between the RIPOR3 gene and the occurrence and development of breast cancer is of great importance in exploring a tumor treatment and early warning strategy aiming at the RIPOR3 pathway.

Disclosure of Invention

In order to solve the above problems, an object of the present invention is to provide a use of a breast cancer molecular marker ripo 3 for preparing a biological product for breast cancer detection, prognosis or treatment, wherein the molecular marker is ripo 3 gene or protein having a sequence represented by NM _001290268.1 or NP _001277197.1 of human ripo 3 as represented by gene ID 140876 of NCBI database.

As used herein, the term ripo 3 (cellular polarization regulating protein 3 of Rho family interaction) refers to the human ripo 3 gene or protein, or homologues thereof, or variant forms thereof having biological activity.

The invention adopts an RT-PCR method to detect the expression of RIPOR3 gene in human breast cancer tissues and tissues beside the cancer, and verifies that the expression of RIPOR3 gene is down-regulated in breast cancer tissues or breast cancer cells.

As used herein, the term "downregulate expression" refers to a decrease in expression of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of the gene expression level measured in the breast cancer tissue of a patient being examined relative to a precancerous normal breast tissue or a healthy human breast tissue, e.g., the gene expression level in the breast cancer tissue is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or less of the normal breast tissue.

One aspect of the present invention provides the use of the molecular marker ripo 3 in the preparation of a kit for the detection or prognosis of breast cancer, in some embodiments, the ripo 3 is human ripo 3.

In some embodiments of the invention, the detecting or prognosing is a disease state determination, a therapy assessment or a metastatic relapse monitoring of breast cancer in a subject based on the level of RIPOR3 expression in a biological sample of the subject, preferably the biological sample is breast cancer tissue or breast cancer cells.

In another aspect, the invention provides a kit for breast cancer detection or prognosis, which comprises a primer pair for specifically amplifying the human RIPOR3 gene, wherein the forward primer is a sequence shown as SEQ ID NO.1, and the reverse primer is a sequence shown as SEQ ID NO. 2; in addition, primers for amplifying internal reference GAPDHThe pair is a forward primer shown as SEQ ID NO.3 and a reverse primer shown as SEQ ID NO. 4. Further, the kit also comprises an RNA extraction reagent, a reverse transcription reagent, and common reagents for PCR reaction such as reverse transcriptase, buffer solution, dNTPs, MgCl₂DEPC water and Taq enzyme, and the like, and may further contain a standard substance and/or a control substance.

In a further aspect of the invention there is provided the use of ripo 3 or an analogue or agonist thereof in the manufacture of a pharmaceutical composition for the prevention or treatment of breast cancer, wherein the analogue or agonist is a variant form thereof having the biological activity of ripo 3.

In some embodiments of the present invention, the pharmaceutical composition comprises an effective amount of ripo 3 or its analog or agonist and a pharmaceutically acceptable carrier, and further comprises other agents for preventing or treating breast cancer.

As used herein, the "effective amount" refers to an amount that produces a function or activity in, and is acceptable by, a mammal (preferably a human). The "pharmaceutically acceptable carrier" refers to a carrier for administration of the therapeutic agent, including various excipients and diluents; the carrier itself is not an essential active ingredient and is not unduly toxic after administration. Such suitable carriers are well known to those of ordinary skill in the art. The pharmaceutically acceptable carrier in the pharmaceutical composition may contain liquids such as water, saline, buffers, and auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances, and the like may also be present. The vector may also contain a cell (e.g., host cell) transfection reagent.

The present invention may employ various methods well known in the art for administering ripo 3 protein, its analogs or agonists, or genes encoding it, or pharmaceutical compositions thereof, to a patient, such as a mammal, preferably a human. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the mode of administration is parenteral.

Alternatively, gene therapy may be used to treat breast cancer, such as by directly administering RIPOR3, an analog or agonist thereof, to the patient by methods such as injection; alternatively, an expression unit, such as an expression vector or virus, carrying ripo 3, an analog or agonist thereof, can be delivered to a target in a manner that results in the expression of active ripo 3, an analog or agonist thereof, depending on the type of agent, as is well known to those of skill in the art.

The pharmaceutical composition of the present invention may further comprise one or more anticancer agents. In particular embodiments, the pharmaceutical composition comprises at least one compound that promotes the expression of RIPOR3 gene and at least one chemotherapeutic agent. Chemotherapeutic agents for use in the pharmaceutical compositions of the present invention include, but are not limited to: DNA-alkylating agents, anti-tumor antibiotic agents, anti-metabolites, tubulin stabilizers, tubulin destabilizers, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-COA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, and molecularly modified viral, bacterial, and exotoxin agents.

In the present invention, "prognosis" refers to the process or outcome of a cancer patient after inhibiting or alleviating tumor growth by surgery, chemotherapy, drug treatment, or a combination thereof. Prognosis can be the inhibition or alleviation of the vital state of breast cancer by surgery, chemotherapy, drug therapy, or a combination thereof, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 years or more after growth. Prognosis can be assessed by detecting a marker, which is one or more genes. The prognostic assessment may be performed by: determining whether the prognosis of the patient is good, or determining the probability of a good or poor prognosis, based on the presence or absence, or increase or decrease, of the marker.

The pharmaceutical compositions of the invention may also be used in combination with other agents for the treatment of breast cancer, and other therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

The pharmaceutical compositions of the present invention may also be administered separately with other therapeutic compounds, either as separate compositions or in different dosage forms than the main active ingredient. A partial dose of the main active ingredient may be administered simultaneously with the other therapeutic compounds, while the other doses may be administered separately. The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.

Has the advantages that:

the inventor finds that the expression of the molecular marker RIPOR3 is obviously down-regulated in the breast cancer tissues of a patient relative to the expression in the tissues beside the cancer, so that RIPOR3 can be used as a marker for auxiliary diagnosis and prognosis of breast cancer and preparation of biological products for detection and/or treatment of the breast cancer. The kit for detecting the breast cancer provided by the invention can be used for auxiliary diagnosis and prognosis evaluation of the breast cancer; in addition, the pharmaceutical composition for treating breast cancer provided by the invention can also be used for clinical application of gene therapy, drug therapy and the like of breast cancer.

Drawings

FIG. 1 shows the relative expression of RIPOR3 in breast cancer tissues and paraneoplastic tissues, wherein the expression level of RIPOR3 in breast cancer tissues is significantly down-regulated relative to normal paraneoplastic tissues.

FIG. 2 shows that the relative expression level of RIPOR3 in the breast cancer cell lines MCF-7 and MDA-MB-453 is significantly lower than that in the normal epithelial cell line MCF-10A of the breast.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

This example demonstrates that RIPOR3 is present in significantly lower amounts in patient breast cancer tissue samples than in paracarcinoma tissue samples.

1. Sampling

Taking 50 cases of tissues beside breast cancer and breast cancer tissues obtained in surgical operation of Shenzhen hospital of Beijing university from 10 months to 12 months in 2013, verifying all specimens by pathological examination, and storing the specimens in a low-temperature refrigerator at-80 ℃ after numbering. RNA was extracted for real-time fluorescent quantitative PCR. All patients reported in paper and specimen collections were approved by the Ethics review Committee of biomedical sciences of Beijing university.

2. Total RNA extraction from tissue samples

By using

Reagent (invitrogen, cat # 15596-:

collecting a sample, freezing the sample in liquid nitrogen, taking out the sample, putting the tissue into a precooled mortar for grinding, and after the tissue sample is powdered:

adding Trizol, and preserving for 5 minutes at room temperature;

adding 0.2mL of chloroform, forcibly oscillating the centrifuge tube, fully mixing the materials, and standing the mixture at room temperature for 5 to 10 minutes;

③ centrifuging at 12000rpm for 15 minutes, sucking the upper water phase (sucking 70%) into another new centrifugal tube, and taking care not to suck the protein material between the two water phases. Moving into a new tube, adding equal volume of pre-cooled isopropanol at-20 ℃, fully reversing and uniformly mixing, and placing on ice for 10 minutes;

fourthly, after 15 minutes of high-speed separation at 12000rpm, the supernatant is carefully discarded, 75 percent DEPC ethanol is added according to the proportion of 1mL/mL Trizol to wash and precipitate (the precipitate is stored at 4 ℃), the precipitate is washed and mixed by shaking, and the mixture is centrifuged at 12000rpm at 4 ℃ for 5 minutes;

fifthly, removing the ethanol liquid, standing for 5 minutes at room temperature to fully air-dry the precipitate, and adding DEPC treated water to dissolve the precipitate;

sixthly, the purity and the concentration of the RNA are measured by a Nanodrop2000 ultraviolet spectrophotometer and are frozen and stored at-80 ℃. RNA quality determination criteria: the OD260/OD280 value of the RNA sample is between 1.7 and 2.2; the total RNA electrophoresis pattern has clear 28S and 18S bands; the electrophoresis pattern after the water bath heat preservation for 1 hour at 70 ℃ has no obvious difference with the pattern before the water bath heat preservation.

3. Reverse transcription to synthesize cDNA

By using

III Reverse transcription of cDNA by Reverse transcription of Transcriptase (Invitrogen, cat # 18080-044), the experimental procedures were performed according to the product instructions, and the specific procedures were as follows:

using a reverse transcription kit, cDNA was synthesized by reverse transcription of l. mu.g of total RNA with reverse transcription buffer. Using a 25. mu.L reaction, 1. mu.g of total RNA was taken for each sample as template RNA. The obtained cDNA was stored in a freezer at-20 ℃ for further use.

4、RT-PCR

Using ABI 7500 type fluorescent quantitative PCR instrument, adopting 2^-ΔΔCtThe method carries out relative quantitative analysis of data.

On-line primer design software was used, with the gene sequence referenced NCBI: RIPOR3 (Gene ID:140876, NM-001290268.1), GAPDH as an internal control, and Invitrogen as a primer design. The specific primer sequences are as follows:

table 1: primer sequences

The operation process is as follows:

table 2: RT-PCR reaction system

Reaction system: by Power

Green PCR Master Mix (Invitrogen, cat # 4367659) was amplified and the experimental protocol was performed according to the product instructions.

The amplification procedure was: pre-denaturation at 95 ℃ for 5min, (denaturation at 95 ℃ for 15sec, Tm annealing for 45sec, extension at 72 ℃ for 35sec) x 40 cycles, the specific Tm is given in Table 1.

And (3) detecting the sample by RT-PCR: after 10-fold dilution of each sample cDNA, 2. mu.L of each sample cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification, respectively. At the same time, the dissolution curve analysis is carried out at 60-95 ℃.

The experimental result shows that the inflection point of the real-time quantitative PCR amplification curve is clear, the overall parallelism of the amplification curve is good, the amplification efficiency of each reaction tube is similar, the limit is flat without rising, the slope of the exponential phase of the curve is larger, and the amplification efficiency is higher; the dissolution curves of the sample amplification products are all unimodal, which indicates that only one amplification product is specifically amplified; according to the relative quantitative formula of qRT-PCR: 2^-ΔΔCtX 100%, the expression levels of the genes in breast cancer tissue and paracancerous tissue were compared. The results show that: the qRT-PCR amplification result is stable, as shown in figure 1, the relative expression level of RIPOR3 gene in breast cancer tissue is obviously lower than that in paracarcinoma tissue, and the difference has statistical significance (P)<0.05); this indicates that RIPOR3 expression is down-regulated in breast cancer tissues of patients.

Example 2 expression of RIPOR3 in breast cancer cell lines

First, a human breast cancer cell line MCF-7, MDA-MB-453 and a normal mammary gland epithelial cell line MCF-10A were cultured, wherein the MDA-MB-453 was cultured in an L15 medium containing 10% fetal bovine serum, and the MCF-7 and the normal mammary gland epithelial cell line MCF-10A were cultured in a DMEM medium containing 10% fetal bovine serum. To the culture solution was added 1% P/S (penicillin-streptomycin mixed solution). Culturing in an incubator at 37 deg.C, 5% CO2, and relative humidity of 90%. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

Then, total RNA in the cells was extracted using a cell RNA extraction kit from QIAGEN and the concentration thereof was measured, and the specific procedures and qPCR were the same as those in example 1. The experiment was performed in 3 replicates with data presented as mean ± sd, and statistical analysis was performed using SPSS18.0 statistical software, and the pairing of breast cancer cell lines with normal breast cell lines using t-test was considered statistically significant when P < 0.05.

As shown in FIG. 2, the relative expression level of RIPOR3 gene in the breast cancer cell lines MCF-7 and MDA-MB-453 was significantly lower than that in the normal epithelial cell line MCF-10A of breast, and the difference was statistically significant (P < 0.05).

Example 3 overexpression of RIPOR3 Gene in Breast cancer cells

1. Cell culture

Human breast cancer cell line MDA-MB-453 was cultured in L15 medium containing 10% FBS and 1% P/S at 37 ℃ in an incubator at 5% CO2 and 90% relative humidity. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

The cells in the culture flask were digested with pancreatin and seeded in 6-well plates to ensure that the number of cells was 2-8X 10⁵Per well, add L15 cell culture medium. The cell density was observed overnight the next day, and transfection was possible at cell densities above 70%.

2. Construction of Gene overexpression vectors

Specific PCR amplification primers were synthesized based on the cDNA sequence of the human RIPOR3 gene, and HindIII and XhoI restriction sites were added to the 5 'primer and 3' primer, respectively. cDNA obtained by blood extraction and reverse transcription of a breast cancer patient is used as an amplification template, the cDNA sequence is inserted into a eukaryotic cell expression vector pcDNA3.1 which is subjected to double enzyme digestion by restriction enzymes HindIII and XhoI, and the obtained recombinant vector pcDNA3.1-1 is connected for subsequent experiments.

3. Transfection

The experiment was divided into three groups: the control group (MDA-MB-453), the negative control group (pcDNA3.1-NC) and the experimental group (pcDNA3.1-1) were transfected with the vector using the liposome 3000, and the specific transfection method was performed as indicated in the specification.

4. qPCR detection of RIPOR3 Gene levels

1) Extraction of Total RNA from cells

The QIAGEN cell extraction kit is used for extracting total RNA in cells, and the specific operation is detailed in the instruction.

2) The specific steps of qPCR amplification were the same as in example 1.

5. Statistical method

The experiments were performed in 3 replicates, the data were expressed as mean ± sd, statistically analyzed using SPSS18.0 statistical software, and the differences between the experimental group and the control group of RIPOR3 genes were determined to be statistically significant when P <0.05 using a t-test.

6. Results

As a result, the RIPOR3 gene expression level of MDA-MB-453 cells was significantly increased in the experimental group compared to the control group, and the difference was statistically significant (P < 0.05).

Example 4 Effect of RIPOR3 Gene on Breast cancer cell proliferation

CCK-8 experiment is adopted to detect the influence of RIPOR3 gene on the proliferation capacity of breast cancer cells.

1. MDA-MB-453 cells were cultured and transfected as in example 3, with the medium changed 6h after transfection and left in the cell incubator overnight.

2. Cells were digested with trypsin, added to L15 cell culture medium and mixed well to suspend the cells, and the cells were counted.

3. The 96-well plate was seeded at 3000/well and 8 multiple wells. A total of 4 plates of 96 wells were plated for 24h, 48h, 72h and 96h 4 detection time points, respectively. .

4. And after 24h, taking out the first 96-well plate, adding 10 mu l of CCK-8 detection solution into each well, continuously putting the 96-well plate into a cell culture box, incubating for about 4h, detecting the absorbance value of each well at the wavelength of 450nm by using an enzyme-labeling instrument, and recording data.

5. And (5) repeating the operation in the step (4) after 48h, 72h and 96h respectively, and finally counting the absorbance values of all time points to make a growth curve graph.

6. Statistical analysis

The experiments were performed in 3 replicates using SPSS18.0 statistical software for statistical analysis, and the differences between the two were considered statistically significant when P <0.05 using the t-test.

7. Results

Results compared with the control, the experimental group has obviously inhibited the proliferation of the cells after being transfected with pcDNA3.1-1, and the difference has statistical significance (P <0.05), which indicates that RIPOR3 is related to the cell proliferation of the breast cancer, and RIPOR3 over-expression can inhibit the proliferation of the breast cancer cells.

Example 5 Breast cancer detection or prognostic evaluation kit

The kit for detecting breast cancer according to the present invention was assembled based on the primer set shown in Table 1 in example 1, and comprised a primer pair (forward primer shown in SEQ ID NO:1 and reverse primer shown in SEQ ID NO: 2) for specifically amplifying the human RIPOR3 gene and a primer pair (forward primer shown in SEQ ID NO:3 and reverse primer shown in SEQ ID NO: 4) for specifically amplifying the housekeeping Gene (GAPDH); it also comprises SYBR Green polymerase chain reaction system, such as PCR buffer solution, SYBR Green fluorescent dye, dNTPs. The PCR buffer solution comprises 25mM KCl and 2.5mM MgCl₂，200mM(NH₄)₂SO₄(ii) a Also included are the normal tissue cDNA of breast: as a negative control, the PCR detection is carried out quantitatively with the sample cDNA to be detected, and the same amount of the sample cDNA to be detected is used in each reaction system. Preferred PCR reaction systems are shown in Table 3:

TABLE 3PCR reaction System

Components	Amount of addition
		SYBR Green polymerase chain reaction system	12.5μL
Upstream primer (10. mu.M)	0.5μL
		Downstream primer (10. mu.M)	0.5μL
Template cDNA	2.0μL
		Adding sterilized distilled water	To 25 μ L

The optimal reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, (denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 45sec, extension at 72 ℃ for 35 sec). times.40 cycles, extension at 72 ℃ for 15 min.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims

1. The application of a breast cancer molecular marker RIPOR3 in preparing biological products for breast cancer detection or treatment is characterized in that the molecular marker is RIPOR3 gene or protein, and the nucleotide or amino acid sequence of the molecular marker is shown as NM _001290268.1 or NP _001277197.1 of human RIPOR3 shown in NCBI database gene ID: 140876.

2. The use according to claim 1, wherein the biological product quantitatively detects the expression level of a molecular marker ripo 3 in breast cancer tissue or breast cancer cells, and the molecular marker ripo 3 is down-regulated in the breast cancer tissue or breast cancer cells of the patient.

3. The use of claim 1 or 2, wherein the biological product is a kit for breast cancer detection.

4. The use according to claim 3, wherein the detection is for the determination of the disease status of breast cancer in a subject, for the assessment of the efficacy of treatment, or for the monitoring of metastatic relapse based on the level of RIPOR3 expression in a biological sample from the subject.

5. The use of claim 4, wherein the biological sample is breast cancer tissue or breast cancer cells.

The use of ripo 3 in the manufacture of a pharmaceutical composition for the prevention or treatment of breast cancer, wherein the pharmaceutical composition comprises ripo 3 and a pharmaceutically acceptable carrier.

7. The use of claim 6, wherein the pharmaceutical composition further comprises an additional agent for the prevention or treatment of breast cancer.