CN109512834B

CN109512834B - Application of hsa-miR-145-5p in treatment of breast cancer

Info

Publication number: CN109512834B
Application number: CN201811332268.XA
Authority: CN
Inventors: 黄来强; 孙春辉; 代小勇
Original assignee: Tsinghua-Berkeley Shenzhen Institute Preparation Office
Current assignee: Tsinghua-Berkeley Shenzhen Institute Preparation Office
Priority date: 2018-11-09
Filing date: 2018-11-09
Publication date: 2021-04-20
Anticipated expiration: 2038-11-09
Also published as: CN109512834A

Abstract

The invention discloses application of hsa-miR-145-5p in treatment of breast cancer, and finds that the content of hsa-miR-145-5p in tumor cells is increased, the drug resistance of cancer cells is remarkably reduced, the activity of the tumor cells is weakened, and the method can be used for treating breast cancer. The combination of hsa-miR-145-5p and the inhibitor of calcineurin can be a means for improving chemotherapeutic drugs and reducing breast cancer mortality, and greatly improves the effect of antitumor drugs in inhibiting tumor cells.

Description

Application of hsa-miR-145-5p in treatment of breast cancer

Technical Field

The invention belongs to the technical field of biology, relates to a tumor molecular marker, and particularly relates to application of hsa-miR-145-5p in treatment of breast cancer and improvement of breast cancer sensitivity to treatment drugs.

Background

The latest global tumor statistical analysis results in 2018 cover 185 national regions and 36 cancers, and the incidence rate and the fatality rate of each cancer in 2018 and the incidence condition of tumors in each region are mainly analyzed. Statistics show that there are about 1819 ten thousand new cancer cases and 960 ten thousand cancer death cases worldwide, with asia accounting for about 50% of the new cancer cases and 70% of the cancer death patients. The final analysis results show that breast cancer is the first "killer" of women (about 210 million new diagnosed female breast cancer cases in 2018 worldwide), and in the globally diagnosed female cancer cases, breast cancer accounts for about 1/4, which is one of the most common malignancies in women.

When regulated by abnormal mechanisms, cells enter a state of malignant proliferation and differentiation, resulting in tumorigenesis. The typical history of malignant tumor growth can be divided into several stages: malignant transformation of single cells → clonal proliferation of transformed cells → local infiltration → distant metastasis. So far, the related pathogenic mechanism of the tumor is not completely clear, and the research on gene and protein signal pathways of tumor cell migration, infiltration and apoptosis has important significance for explaining the tumor pathogenic mechanism and preventing and treating the tumor. Normal apoptosis refers to the autonomous and orderly death of cells controlled by genes to maintain homeostasis, and is a process strictly controlled by multiple genes, and the genes are well conserved among species, such as Bcl-2 family, caspase family, oncogenes such as C-myc, and cancer suppressor gene P53. The disturbance of the apoptotic process may be directly or indirectly related to the development of many diseases, such as tumors, autoimmune diseases, etc.

MicroRNA (miRNA) is a non-coding single-stranded micro RNA molecule which is coded by endogenous genes and has the length of about 22 nucleotides, plays a role in regulating various vital activities in cells, including development, organ formation, cell proliferation and apoptosis, fat metabolism and the like, and has an important gene expression regulation function. This complex regulatory network can regulate the expression of multiple genes by a single miRNA or can finely regulate the expression of a gene by a combination of several mirnas. The action mechanism of the existing known miRNA is the following points that (1) translation inhibition is performed, and the miRNA is not completely complementary with target mRNA, so that the miRNA can inhibit target gene expression at the protein translation level; (2) degradation of mRNA, complete complementarity (or nearly complete complementarity) of the miRNA to the target site, binding of the miRNA causing degradation of the target mRNA; (3) transcription regulation, miRNA influences CpG island methylation of gene promoters, and the target gene is directly regulated and controlled at the transcription level. Due to the characteristics of high conservation, space-time specificity, stability, tissue specificity and the like, the miRNA is superior to other biomarkers such as protein, DNA fragments and the like, and plays more and more important roles in disease pathogenesis research, early diagnosis, individualized treatment, prognosis and the like.

Chemotherapy is one of the most important means of treating malignant tumors, however, resistance of tumor cells to chemotherapeutic drugs often ultimately leads to failure of chemotherapy. In vivo and in vitro researches find that the molecular mechanism of tumor drug resistance is complex and mainly comprises the following steps: activation of drug transporter, excluding intracellular drug from body; reduce drug activation or enhance intracellular drug detoxification; change of drug target, repair and enhancement of damaged target, and apoptosis inhibition or cell cycle arrest. Breast cancers are generally classified into four molecular subtypes, Luminal type A (ER +/PR +, HER-2-), Luminal type B (ER +/PR +, HER-2+), HER-2+ (ER-/PR-/HER-2+), and Basal-like (ER-/PR-/HER-2-), wherein Luminal A, comprises about 50-60%; LuminalB, accounting for 10-20%. The clinical treatment response and survival time of breast cancer of different molecular subtypes are different, and the clinical attention is more and more attracted at present. Tamoxifen (Tamoxifen) is used for treating advanced breast cancer and ovarian cancer, clinically treating breast cancer, the effective rate is generally 30%, and the curative effect of estrogen receptor positive (ER +) breast cancer patients is better (49%). However, recent studies have shown that the incidence of adverse reactions in the reproductive system is high and that about half of patients with ER-positivity develop a resistant phenotype. Factors that contribute to resistance to tamoxifen include (1) the pharmacological mechanism of tamoxifen; (2) ER α expression deletion, mutation or post-translational modification; (3) imbalance of co-regulatory proteins and changes in different signal transduction pathways. Drug resistance limits the treatment of breast cancer patients, and miRNA-based gene therapy provides another attractive cancer suppression approach.

Calcineurin (CaN) belongs to the serine/threonine phosphatase family and is the only Ca receptor discovered to date²⁺And calcineurin-regulated phosphatases, cytoplasmic Ca²⁺Regulation when intracellular Ca²⁺When the concentration is increased, the activity of CaN is promoted, and the phosphorylation level of related proteins in cells is regulated and controlled. At present, many studies have been made to explore its functions, such as: medina found Ca in its 2015 study²⁺Regulating autophagy by regulating a signal path of CaN and TFEB; hitesh Soni, 2016, describes the regulation of Ca²⁺Signal pathway activation of Ca²⁺The CaN/NFATc1 and FasL/Fas signaling pathways, are capable of increasing neonatal mesangial apoptosis. Regarding the role of CaN in neurodegenerative diseases, Maulilio John K found in studies to increase the regulator Ca of plasma membrane CaN²⁺-ATPase activity, activating the CaN/NFAT signaling pathway, promoting the death of nerve cells and glial cells. 2016 on Nat Med: CaN in epithelial cells controls the development of microbe-dependent intestinal tumors, and raises the question whether CaN and NFAT have carcinogenic effects in cells, and through verification, CaN and NFAT promote intestinal tumorigenesis by regulating the functions of cancer stem cells of mice, and are associated with increased activation and death in human colorectal cancer. In the research on the effect of CaN on breast cancer, Zhao researches find that quercetin (Qu) inhibits the activity of CaN, further influences the subcellular localization of vascular endothelial growth factor receptor 2(VEGFR2) and finally inhibits human milkAngiogenesis of adenocarcinoma xenografts in nude mice. Therefore, the CaN is involved in regulating important cell life processes, such as autophagy, apoptosis and the like.

Mirnas play an important role in a range of physiological and pathological processes, and CaN play an important role in cellular activities. However, the mechanism of how miRNA regulates CaN promote drug resistance of breast cancer is not clear. Therefore, the research and discovery of the miRNA angle for the first time through the miRNA show that the miRNA CaN promote the generation of breast cancer to weaken the chemotherapy resistance through the regulation of CaN, provide an important theoretical basis for the clinical medication of the breast cancer, and have a relatively high clinical application prospect.

Disclosure of Invention

One purpose of the invention is to provide application of hsa-miR-145-5p or/and hsa-miR-145-5p mimics in preparation of products for treating or assisting in treating tumors.

The invention also aims to provide application of hsa-miR-145-5p or/and hsa-miR-145-5pmimics in preparation of a product for improving tumor drug sensitivity.

The invention further aims to provide application of hsa-miR-145-5p or/and hsa-miR-145-5p mices in preparation of a product for inhibiting expression of the CaN protein.

The technical scheme adopted by the invention is as follows:

application of hsa-miR-145-5p or/hsa-miR-145-5 p mices in preparation of products for treating or assisting in treating tumors.

Further, the tumor is breast cancer.

Application of hsa-miR-145-5p or/and hsa-miR-145-5p mimics in preparation of products for improving tumor drug sensitivity.

Further, the tumor is breast cancer.

Further, the tumor drug sensitivity is the sensitivity of breast cancer to tamoxifen.

And (3) application of hsa-miR-145-5p or/and hsa-miR-145-5p mics in preparation of products for inhibiting expression of CaN proteins.

Further, the CaN protein expression inhibition is the CaN protein expression inhibition in tumor cells.

Further, the tumor cell is a breast cancer cell.

Further, the breast cancer cells comprise MCF7 and MDA-MB-231.

Further, the hsa-miR-145-5p mimics comprise a sequence shown in SEQ ID NO. 2.

The invention has the beneficial effects that:

the invention discovers that the content of hsa-miR-145-5p in tumor cells is increased, the drug resistance of cancer cells is obviously reduced, the activity of the tumor cells is weakened, and the tumor cells can be used for treating breast cancer. The combination of hsa-miR-145-5p and the inhibitor of calcineurin can improve chemotherapeutic drugs and reduce breast cancer mortality, and greatly improve the effect of antitumor drugs in inhibiting tumor cells.

Drawings

FIG. 1.qPCR screens for miRNA that are underexpressed in MCF7 and MDA-MB-231 cells while being highly expressed in HeLa cells.

FIG. 2 is a graph of the detection of the transfection efficiency of miRNA imic transfected MCF cells, an A graph is a fluorescence detection graph of a transfection indicator transfected MCF7 cells, and a B graph is a fluorescence detection graph of a transfection indicator transfected MDA-MB-231 cells.

FIG. 3, different miRNA regulate the expression of CaN protein in breast cancer cells MCF7, A.hsa-miR-1271-5pmimic promotes the expression of CaN in MCF7 cells, and hsa-miR-1271-5inhibitor inhibits the expression; and B, inhibiting protein expression of CaN in MCF7 cells by using hsa-miR-145-5p micic, and promoting the expression of hsa-miR-145-5p micic inhibitor.

FIG. 4MTT assay shows the survival of MCF7 cells in different treatment groups, in which MCF 7-micnc represents MCF7 cell transfection micic negative control, and MCF7-inhibitor NC represents MCF7 cell transfection inhibitor negative control.

Detailed Description

The present invention will be further described with reference to the following examples.

The application mainly explores how miRNA can regulate the function of calcineurin in breast cancer cells. The research is mainly carried out from the following aspects: (1) selecting 10 miRNAs for inhibiting CaN expression through database targetscan and MiRDA prediction, (2) screening 2 miRNAs meeting conditions through a qPCR experiment, (3) transfecting an miRNA transfection indicator with lipo2000, and observing a MCF7 cell transfection effect through a fluorescence microscope, (4) carrying out cell transfection and western blot detection, and finally obtaining a condition meeting hsa-miR-145-5p, synthesizing the mimicr of a miRNA site hsa-miR-145-5p, inhitor and a corresponding negative control, and (5) finding out MTT cell activity experiments, and improving the sensitivity of an antitumor drug tamoxifen after the MCF7 cell transfects the mimicr of hsa-miR-145-5 p. In conclusion, the application describes that miRNA is involved in regulating drug sensitivity of breast cancer cells MCF7 through regulating target genes.

In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.

Example 1

1 materials and methods

1.1 major cell lines: MCF 7.

1.2qPCR experiments

1.2.1 extraction of Total cellular RNA

Collecting cells, adding Trizol, blowing, mixing, and standing at room temperature for 5 min. Centrifuging at 4 deg.C at 10000 Xg for 10min, collecting supernatant, adding 0.2ml chloroform, shaking vigorously for 30s, and standing for 3 min. Centrifuge at 4 ℃ for 12000g 15min, pipette the supernatant into a fresh DEPC-treated 1.5ml EP tube, add 0.5ml isopropanol, mix gently. Centrifuging at 4 deg.C, 12000g × 10min, discarding the supernatant, adding 1ml of 75% ethanol (ice-cold), shaking, and washing the precipitate thoroughly. Centrifugation is carried out at 4 ℃ for 12000g × 5 min. Discard the supernatant, centrifuge briefly, carefully aspirate and discard the supernatant. An appropriate amount (20. mu.l) of DEPC (RNase free) was added to dissolve the RNA.

1.2.2qPCR experiments

(1) Synthesizing a primer of the corresponding miRNA according to the prediction result,

(2) reverse transcription is performed. Reaction solution system: 1 mu g of total RNA, 10 miRNA reverse transcription primers, water to 12.5 mu l, and mixing evenly. Incubating at 85 deg.C for 5min, immediately placing on ice,

(3) preparing another reaction solution: 2.0. mu.l of 10mM dNTP, 0.5. mu.l of RNase inhibitor, 0.5. mu.l of U6 reverse transcription primer, 4.0. mu.l of 5 XBuffer, 0.5. mu.l of M-MLV,

(4) adding the solution 3) into the solution 1), uniformly mixing, and keeping the temperature at 30 ℃ for 10 min; incubating at 42 deg.C for 5 min; incubating the mixture for 10min at the temperature of 85 ℃,

(5) and (5) carrying out quantitative PCR detection.

1.3 transfection of cells

1.3.1 transfection of miRNA transfection indicators

Inoculation 1X10⁵Cells were plated in 24-well plates and cultured overnight. Mixing TRACER^TMFluorecent Oligo and lipo2000 were mixed with serum-free DMEM and incubated at room temperature for 15 min. The mixture was added to the cells and cultured for 12 h. DMEM with 10% serum was added again and the culture was continued for 48 h. And (4) observing under an inverted fluorescence microscope.

1.3.2 Mimic of miRNA transfected by cells

And synthesizing hsa-miR-1271-5p micic and hsa-miR-145-5p micic. Inoculation 1X10⁵Cells were plated in 24-well plates and cultured overnight. The cells reached 50%, mix the imic with lipo2000 reagent in proportion in serum free DMEM and incubate for 15min at room temperature. The mixture was added to the cells and cultured for 12 h. DMEM with 10% serum was added again and the culture was continued for 48 h.

1.4Westernblot experiment

1.4.1 protein extraction

The MCF7 cells cultured for 48h after transfection are digested by pancreatin, collected, centrifuged, washed by PBS for three times, added with lysate and protease inhibitor, incubated on ice for 30min, boiled at 100 ℃ for 30min, and stored at-20 ℃ for a short time.

1.4.2SDS-PAGE gel electrophoresis

12% of separation glue and 5% of concentrated glue are prepared. Each hole is added with 10 mul of protein sample and protein Marker respectively, and the current is regulated to 30mA for 30 min. After the protein sample is separated into the gel, the current is adjusted to 50mA for 1h, and the electrophoresis is stopped when the indicator-bromophenol blue in the protein sample approaches the bottom of the gel.

1.4.3 protein transfer

The PVDF membrane is soaked in methanol for 5min, and the filter paper is soaked in a membrane transfer buffer solution. The gel was removed and the gel containing the protein of interest was cut off against Marker. According to the requirements of the semi-dry transfer film: the filter paper-PVDF-gel film-filter paper are laid in sequence, 25V and 200mA are carried out, and the transfer printing is carried out for 1.5 h.

1.4.4 antibody incubation

Taking out the PVDF membrane, soaking in 5% fat-free milk, and sealing for 1 h; PBST is washed for 3 times, PVDF membrane containing target protein and beta-actin is cut according to a protein Marker, anti-CaN antibody and anti-actin antibody are respectively incubated, the membrane is slowly swung at 4 ℃ on a shaking table, and the membrane is incubated overnight; PBST was washed 3 sides, anti-rabbitsecondary antibody was added, and incubation was performed at room temperature for 1 h.

1.4.5 luminescence and imaging analysis

PBST was washed 3 times, ECL luminophore was added, the membrane was placed in a gel imager, software imaged and recorded for analysis.

1.5MTT assay

1.5.1 transfection of cells

Seeding cells 1X10⁴Duplicate and control wells were set in 96-well plates and incubated overnight. The cells reach 50%, mix miRNA mix (mimic), mix negative control of mimic, inhibitor negative control with lipo2000, perform cell transfection, and continue to culture for 48 h.

1.5.2 tamoxifen treatment

Tamoxifen was diluted in serum free DMEM, added to the wells and cultured for 24 h.

1.5.3 cell death assays

(1) Adding 20 mu L of prepared MTT into each hole, incubating for 4h in a dark place,

(2) carefully discarding the culture medium, slowly adding 150 μ L DMSO into each well, shaking the shaker at low speed for 15min,

(3) setting the wavelength of an enzyme-linked immunosorbent assay (ELIASA) to be 490nm, measuring the light absorption value of each pore on an enzyme-linked immunosorbent assay (ELISA) monitor, and recording the result.

2 results and analysis

2.1 prediction of target miRNA

Selecting a species: human, gene of interest: PPP3CA was first screened using the Targetscan website, based on the factors considered: calculating the type of species region, the conservation among species and the region enriched by AU in 3' UTR to obtain miRNA targets which are ranked at the top and possibly have functions, and predicting to obtain miRNA which is possibly inhibited from CaN expression

2.2qPCR screening of target miRNAs

It is known that the expression level of CaN protein in the breast cancer cells MCF7 and MDA-MB-231 is higher than that in the normal breast cells MCF10A, and the CaN in the HeLa cells is lower than that in the normal breast cells MCF 10A. The miRNA has the functions of identifying target mRNA by means of base complementary pairing, and guiding a silencing complex to degrade the target mRNA or inhibiting the translation of the target mRNA according to different complementary degrees. Therefore, according to the function of miRNA, miRNA is selected to be low expressed in MCF7 and MDA-MB-231 cells, and high expressed in HeLa cells.

The results are shown in figure 1, hsa-miR-1271-5p and hsa-miR-145-5p meet the conditions, are low expressed in MCF7 and MDA-MB-231 cells, and are high expressed in HeLa cells; while other miRNAs do not meet this condition.

2.3 expression level of CaN protein regulated by hsa-miR-145-5p in tumor cells

The miRNA mature body sequences (5'-3') of the hsa-miR-1271-5p micic and the hsa-miR 145-5p micic are as follows:

hsa-miR-1271-5p mimic：CUUGGCACCUAGCAAGCACUCA(SEQ ID NO:1)；

hsa-miR-145-5p mimic：GUCCAGUUUUCCCAGGAAUCCCU(SEQ ID NO:2)。

1) transfection of miRNA transfection indicator: inoculation 1X10⁵Cells were plated in 24-well plates and cultured overnight. Mixing TRACER^TMFluorecent Oligo and lipo2000 were mixed with serum-free DMEM and incubated at room temperature for 15 min. The mixture was added to the cells and cultured for 12 h. DMEM with 10% serum was added again and the culture was continued for 48 h. And (4) observing under an inverted fluorescence microscope. The detection result is shown in FIG. 2, wherein, the A picture is a fluorescence detection picture of the transfection indicator transfected cell MCF7, and the B picture is a fluorescence detection picture of the transfection indicator transfected cell MDA-MB-231.

2) Mix micic and lipo2000, transfect to MCF7 cells, and culture for 48 h. Collecting cells, extracting proteins, and verifying the influence of hsa-miR-1271-5pmimic, hsa-miR-145-5pmimic, hsa-miR-1271-5p inhibitor and hsa-miR-145-5p inhibitor on the expression of the CaN protein by using western blot.

The detection result is shown in figure 3, hsa-miR-1271-5pmimic promotes the expression of CaN in MCF7 cells, and hsa-miR-1271-5inhibitor inhibits the expression of CaN in MCF7 cells; the hsa-miR-145-5p micic inhibits protein expression of the CaN in the MCF7 cell, and the hsa-miR-145-5p micic inhibitor promotes the expression of the CaN in the MCF7 cell. According to the miRNA function description, hsa-miR-145-5p is selected as a target miRNA for treating tumors.

2.4 Effect of hsa-miR-145-5p on drug resistance of tumor cells

MCF7 cells are transfected with hsa-miR-145-5pmimic, micic negative control, hsa-miR-145-5pinhibitor and inhibitor negative control, and cultured for 48 h; tamoxifen was diluted to 0 μ M, 2 μ M, 4 μ M, 6 μ M, 8 μ M, 10 μ M in serum-free DMEM, cells were added, cultured for 24h, and untreated with drug as a control group (0 μ M). MTT was developed and statistical analysis was performed for viability of MCF7 cells.

The detection result is shown in figure 4, and it can be seen that, at 6 μ M tamoxifen concentration, the activity of MCF7 cells transfected with hsa-miR-145-5pmimic is significantly lower than that of MCF7 cells transfected with hsa-miR-145-5pinhibitor, and the death number of MCF7 cells transfected with hsa-miR-145-5p imic is greater than that of MCF7 cells transfected with hsa-miR-145-5p inibitor, which indicates that hsa-miR-145-5p imic can improve the sensitivity of tumor cells MCF7 to anti-tumor drug tamoxifen and improve the treatment effect on tumor cells. The fact that the hsa-miR-145-5p imic increases the sensitivity of MCF7 to the anti-tumor drug tamoxifen through a signal path for reducing the expression of the CaN protein is shown.

Although differential expression profiles of mirnas have been reported in breast cancer, mirnas that really have a correlation with the target gene PPP3CA of the CaN that one wants to detect and that act on tumor cells have not been discovered. According to the invention, the regulation and control relation between 3 miRNAs and CaN of breast cancer cells MCF7, MDA-MB-231 and HeLa is found through qPCR, and further through detection of western blot, hsa-miR-145-5p is found to have a direct regulation and control relation to CaN protein expression in MCF7 cells (see figure 3). Therefore, mimic and inhibitor (synthesized by bio-corporation) of hsa-miR-145-5p are synthesized, and the influence on apoptosis, proliferation and the like of MCF7 is further detected. In the above MTT experimental results, although the cells start to die more at 6 μ M tamoxifen concentration, the value of the activity of MCF7 transfected with hsa-miR-145-5pinhibitor is obviously greater than that of MCF7 transfected with mimic (see FIG. 4), which indicates that the inhibitor of hsa-miR-145-5p increases the resistance of MCF7 cells to tamoxifen, and the sensitivity of MCF7 to tamoxifen, namely the level of hsa-miR-145-5p is increased, the expression of CaN protein is reduced, and the death of MCF7 cells CaN be promoted. The results suggest that the mimic and the inhibitor of hsa-miR-145-5p are possibly involved in aspects such as tumor cell proliferation and invasion through the regulation and control of CaN. The invention helps to take hsa-miR-145-5p and a downstream signal channel regulated by the hsa-miR-145-5p as a target for tumor treatment by deeply describing a regulation mechanism of the hsa-miR-145-5p on a downstream target gene CaN, and provides a new way for clinical tumor treatment.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

<110> preparation office of the institute of Qinghua-Berkeley Shenzhen

Application of <120> hsa-miR-145-5p in treatment of breast cancer

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> RNA

<213> Artificial sequence

<400> 1

cuuggcaccu agcaagcacu ca 22

<210> 2

<211> 23

<212> RNA

<213> Artificial sequence

<400> 2

guccaguuuu cccaggaauc ccu 23

Claims

The application of a mimic of hsa-miR-145-5p or/and hsa-miR-145-5p in the preparation of a product for improving the sensitivity of tumor drugs; the tumor is breast cancer; the tumor drug sensitivity is the sensitivity of breast cancer to tamoxifen;

wherein, the sequence of the mimic of the hsa-miR-145-5p is shown in SEQ ID NO. 2.