Application of product for detecting FBF1 gene or FBF1 protein in preparation of breast cancer cell stemness characteristic detection tool

Abstract

The invention relates to the technical field of biology, and particularly discloses application of a product for detecting an FBF1 gene or an FBF1 protein in preparation of a breast cancer cell stemness characteristic detection tool, the coding sequence of the FBF1 gene is shown as SEQ ID NO.1, and the amino acid sequence of the FBF1 protein is shown as SEQ ID NO.2. The invention further discloses a kit for detecting breast cancer cell stemness characteristics. The FBF1 gene or FBF1 protein provided by the invention can be used as a detection marker for breast cancer cell stemness characteristics, and FBF1 is highly expressed in breast cancer and can be used for preparing a tool for detecting breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a product for detecting FBF1 gene or FBF1 protein in preparing a tool for detecting stemness characteristics of breast cancer cells. Background Art

[0002] Breast cancer is the most common malignant tumor in women, which seriously affects women's physical and mental health. When malignant lesions occur, breast cancer often metastasizes to distant sites, such as bones, brain and lungs, and eventually leads to the patient's death.

[0003] Tumor metastasis is the main cause of treatment failure and death in most patients with malignant tumors in clinical practice. At the same time, tumor metastasis is a comprehensive, multi-stage, multi-step complex process. In the early stage, most tumor cells can invade blood vessels, survive and overflow blood vessels to reach the expected metastatic location. However, in the late stage of tumor metastasis, less than 2% of tumor cells can grow in the target organ and form micrometastases, and only about 0.02% of cells can eventually develop into tumor metastases. Therefore, finding this group of cells and in-depth exploration of the molecular mechanism of their function is of great significance for the elucidation of the mechanism of tumor malignancy and the development of related anti-tumor drugs.

[0004] Studies have shown that tumor cells that can eventually achieve metastasis must have certain intrinsic specificity, be able to adapt to the changes in the microenvironment during metastasis and the new environment of the target organ, and complete self-replication, growth and proliferation in the new environment. According to current research, cancer stem cells (CSCs) have this specificity. At the same time, the "seed and soil" theory of tumor metastasis mechanism believes that high-incidence tissues provide a microenvironment suitable for cancer cell proliferation, that is, "soil". After screening, the cancer cell "seeds" that are transferred here can form tumors through growth and proliferation. With the rise of CSCs research and the successful identification and separation of solid tumor stem cells such as breast cancer and prostate cancer, there is a lot of evidence showing that CSCs are the group of "seed" cells we are looking for. These cells can both escape the recognition of the body's own immune system and resist external killing, and can eventually form tumor metastasis sites under the induction of certain factors. It can be seen that CSCs are closely related to tumor metastasis. Therefore, the detection of breast cancer cell stemness characteristics is of great significance, which helps to accurately evaluate the occurrence and development of cancer, is conducive to the formulation of treatment measures, and improves the prognosis of patients. Therefore, it is necessary to develop new approaches to detect the stemness characteristics of breast cancer cells. Summary of the invention

[0005] In order to detect the stemness characteristics of breast cancer cells, the present invention provides the use of a product for detecting the FBF1 gene or FBF1 protein in the preparation of a tool for detecting the stemness characteristics of breast cancer cells. The FBF1 gene or FBF1 protein provided by the present invention can be used as a marker for detecting the stemness characteristics of breast cancer cells. FBF1 is highly expressed in breast cancer and can be used to prepare a tool for detecting breast cancer.

[0006] The present invention provides an application of a product for detecting FBF1 gene or FBF1 protein in preparing a tool for detecting stemness characteristics of breast cancer cells. The coding sequence of the FBF1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FBF1 protein is shown in SEQ ID NO.2.

[0007] The FBF1 gene or FBF1 protein provided by the present invention can be used as a detection marker for the stemness characteristics of breast cancer cells. FBF1 is highly expressed in breast cancer and can be used to prepare a tool for detecting breast cancer.

[0008] Furthermore, the product for detecting the FBF1 gene or FBF1 protein includes a reagent for detecting the expression level of the FBF1 gene or FBF1 protein.

[0009] Furthermore, the reagent for detecting the expression level of the FBF1 gene includes primers for amplifying the FBF1 gene shown in SEQ ID NO.3 to SEQ ID NO.4.

[0010] Furthermore, the tool includes a nucleic acid capable of binding to the FBF1 gene or a substance capable of binding to the FBF1 protein.

[0011] Furthermore, the substance capable of binding to the FBF1 protein includes an antibody that specifically binds to the FBF1 protein.

[0012] The present invention also provides an interference vector containing the shRNA that interferes with the FBF1 gene. The interference vector of the shRNA is obtained by annealing the shRNA shown in SEQ ID NO.5 to generate a double-stranded product and then connecting it with the pLV-H1-EF1α-puro vector.

[0013] The present invention also provides a lentivirus containing the interference vector, which is obtained by packaging the shRNA interference vector into a lentivirus.

[0014] The present invention also provides the use of an FBF1 gene inhibitor in the preparation of a drug for treating breast cancer, wherein the FBF1 gene inhibitor has the shRNA interfering with the FBF1 gene as the only effective ingredient.

[0015] Furthermore, the drug has the following effects:

[0016] Inhibit the stemness of breast cancer cells;

[0017] Reduced the side population cell proportion and sphere-forming ability of breast cancer cells.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The FBF1 gene or FBF1 protein provided by the present invention can be used as a detection marker for the stemness characteristics of breast cancer cells. FBF1 is highly expressed in breast cancer and can be used to prepare a tool for detecting breast cancer.

[0020] The FBF1 in the present invention is highly expressed in breast cancer. Through side population experiments, spheroidization experiments, qRT-PCR experiments and Western Blot experiments, it was found that silencing the FBF1 gene can inhibit the stemness of breast cancer cells, while overexpressing the FBF1 gene can increase the proportion of side population cells, enhance the cell spheroidization ability, and promote the expression of tumor cell stemness markers such as SOX2 and OCT4. Molecular mechanism studies have found that FBF1 can promote the stemness characteristics of breast cancer cells through the PI3K / AKT / SOX2 signaling pathway.

[0021] The present invention discovered for the first time that the expression of FBF1 is related to the stemness characteristics of breast cancer cells. The stemness characteristics of breast cancer cells can, to a certain extent, reflect the possibility of metastasis and treatment resistance in breast cancer patients, providing new ideas and new solutions for the treatment of breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 is the expression level of FBF1 in normal tissues and breast cancer tissues.

[0024] Figure 2 To investigate the effect of overexpression of FBF1 on the stemness of breast cancer cells;

[0025] In the figure, A is a side population experiment to detect the effect of overexpression of FBF1 on the proportion of side population cells in breast cancer T47D cells;

[0026] B is a statistical graph of the side population cell ratio of breast cancer T47D cells detected by overexpression of FBF1 in the side population experiment;

[0027] C is a microscopic image of the spheroidization experiment to detect the effect of overexpression of FBF1 on the spheroidization of breast cancer cells;

[0028] D is a statistical graph showing the effect of overexpression of FBF1 on the spheroidization ability of breast cancer cells in a spheroidization experiment;

[0029] E is a qRT-PCR experiment to detect the effect of overexpression of FBF1 on the expression of transcription factors that maintain the stemness of tumor cells;

[0030] F is a Western Blot experiment detecting the effect of overexpression of FBF1 on the expression of transcription factors that maintain the stemness of tumor cells.

[0031] Figure 3 To investigate the effect of silencing FBF1 on the stemness of breast cancer cells;

[0032] In the figure, A is a qRT-PCR experiment to detect the effect of silencing FBF1 on the expression of transcription factors that maintain the stemness of tumor cells;

[0033] B is a Western Blot experiment to detect the effect of silencing FBF1 on the expression of transcription factors that maintain the stemness of tumor cells;

[0034] C is a side population cell experiment to detect the effect of silencing FBF1 on the proportion of side population cells in breast cancer MDA-MB-231 cells;

[0035] D is a statistical graph showing the effect of silencing FBF1 on the proportion of side population cells in breast cancer MDA-MB-231 cells in a side population experiment;

[0036] E is a microscopic image of the spheroidization experiment to detect the effect of silencing FBF1 on the spheroidization of breast cancer cells;

[0037] F is the statistical graph of the spheroidization ability of breast cancer cells detected by silencing FBF1 in the spheroidization experiment.

[0038] Figure 4 Analysis of FBF1-regulated PI3K / AKT signaling pathway;

[0039] In the figure, A is the heat map analysis of differentially expressed genes;

[0040] B is a bar chart of KEGG pathway classification;

[0041] C is the KEGG pathway bubble diagram;

[0042] D is the biological process of differentially expressed genes in GO enrichment analysis;

[0043] E is the molecular function of differentially expressed genes in GO enrichment analysis;

[0044] F is the expression of genes analyzed by qRT-PCR;

[0045] G is Western blot analysis of the effect of FBF1 on the expression of p-PI3K and p-AKT.

[0046] Figure 5 The effect of PI3K inhibitor on the promotion of breast cancer cell stemness by overexpression of FBF1;

[0047] In the figure, A is a side population experiment to detect the effect of overexpression of FBF1 and PI3K inhibitor on the proportion of side population cells in breast cancer T47D cells;

[0048] B is a statistical graph showing the effects of overexpression of FBF1 and PI3K inhibitor on the proportion of side population cells in breast cancer T47D cells.

[0049] C is a microscopic image of the spheroidization experiment to detect the effects of overexpression of FBF1 and PI3K inhibitor on the spheroidization of breast cancer cells;

[0050] D is a statistical graph showing the effects of overexpression of FBF1 and PI3K inhibitor on the spheroidization ability of breast cancer cells detected by spheroidization experiment.

[0051] Figure 6 The effect of FBF1 on breast cancer tumor growth and lung metastasis in mice;

[0052] In the figure, A shows the effect of overexpression of FBF1 and PI3K inhibitor on breast cancer tumor size in mice;

[0053] B is a statistical graph showing the effect of overexpression of FBF1 and PI3K inhibitor on breast cancer tumor volume in mice;

[0054] C is a statistical graph showing the effect of overexpression of FBF1 and PI3K inhibitor on breast cancer tumor weight in mice;

[0055] D is the H&E staining of breast cancer lung metastasis in mice overexpressing FBF1 and PI3K inhibitor;

[0056] E shows the effect of overexpression of FBF1 and PI3K inhibitor on the number of lung metastases of breast cancer in mice;

[0057] F shows the effect of overexpression of FBF1 and PI3K inhibitor on the proportion of lung metastasis area of ​​breast cancer in mice. DETAILED DESCRIPTION

[0058] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0059] Example 1: Use of a product for detecting FBF1 gene or FBF1 protein in preparing a tool for detecting the stemness characteristics of breast cancer cells.

[0060] 1. Experimental Materials and Methods

[0061] 1. Experimental Materials

[0062] Human breast cancer cells T47D and MDA-MB-231 were obtained from ATCC.

[0063] 2. Cell culture

[0064] T47D cells were cultured in high-glucose DMEM medium (BI) containing 10% fetal bovine serum (Gibco) and 1% NEAA (Gibco) in a 37°C incubator under 5% carbon dioxide concentration. MDA-MB-231 cells were cultured in L15 medium containing 10% fetal bovine serum in a 37°C incubator.

[0065] 3. Recombinant plasmid construction

[0066] Using mRNA extracted from T47D cells as a template, first reverse transcription was performed into cDNA, and the reaction system is shown in Table 1. Reaction conditions: 42°C for 30 min, 85°C for 5 min, and 4°C forever.

[0067] Table 1 Reaction system

[0068] Components Volume Total RNA 50ng-5μg <![CDATA[Anchored Oligo-dT 18 (0.5μg / μl)]]> 1μl 2×Ts Reaction Mix 10μl TransScript RT / RI Enzyme Mix 1μl RNase-free water to 20μl

[0069] Then, the coding region sequence of the FBF1 gene was amplified by PCR using FBF1-clone-F shown in SEQ ID NO.3 and FBF1-clone-R shown in SEQ ID NO.4 as primers, and restriction sites Nhe I and Xba I were introduced during amplification.

[0070] SEQ ID NO.3:

[0071] 5'-CGGCTAGCGCCACCATGGCACCAAAAACCAAGAA-3'.

[0072] SEQ ID NO. 4: 5'-GCTCTAGATCAGGCTGAATGAGATGTCAAA-3'.

[0073] The coding region sequence of the amplified FBF1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0074] SEQ ID NO.1:

[0075] ATGGCACCAAAAACCAAGAAAGGATGTAAAGGCTCCATTGATGATTTTCT

[0076] TGGTGACCTTCTAGGGGATGATATGACACTACCTGAGAAGCCTGTTAAACT

[0077] AGCTTCACATACCAGAGACACCACAGGTGTATCTCAGATGTTCCCTTCTTC

[0078] AAAGGCGAGAACAAAGTCCCTCCTGGGTGATGATGTCTTCAGCACCATGG

[0079] CAGGCCTGGAAGAAGCTGATGCTGAGGTTTCAGGTATCTCAGAGGCAGA

[0080] CCCACAGGCTCTGCTCCAGGCCATGAAGGACCTGGACGGCATGGATGCTG

[0081] ATATCTTAGGTCTGAAGAAATCTAATTCAGCCCCTAGCAAAAAAGCTGCA

[0082] AAGGACCCTGGGAAAGGAGAGCTGCCCAACCACCCCAAGCCTGCAGGG

[0083] GGTGCCATTCCCACCAAGAAGTCACTTCCGTCTCCCAGCAGCTCTGGGCA

[0084] TCAGAACAGGAGGTTTTCCTCTGAAGACTTGGAAGACCCATTGAGAGGA

[0085] CTTCTCTCCTATGATGAAGGAGGAATCACCAAGCAGCCGCCTGTGACACA

[0086] GAGTAAAACAGCTTCTGACAAGAGCCCCAGCACAGTGAGAGATCAAGGT

[0087] CCCTCTATTCCTCTAACTCCTGGGGACACCCCCATCCGAAAAAAAGAAGA

[0088] ATTGTTGTTTGATGATGGGGATGACATCATGGCCACCTTGGGGTTTGGAGA

[0089] CAGCCCCAAAGCAGAGAAGAGGCAGATAGGAGACCAGGAAGGGCCTCG

[0090] CCCTGCTCGCTCCACGCTGGATGAGCTGCTGGGTCGAGGCATGGCCACCA

[0091] AACTCCTGGCCCGCCCGGGCACCGGGGAGCACAGGGAGTTCAAGCTAGA

[0092] CAAGAAGTACCAGAGGCCACAGGACAGTGAAGATATGTGGGGTGACGAG

[0093] GACTTCACCTTTGGAGCCTATCAGCCCACTGTGGTCTCCTCTGAGGGCCG

[0094] GCAGTCCCGCCGGCAGTCTGTCAGTAGGTTCTTCGCAGACAGTGGCGCAG

[0095] ACCCCAAGGGAGAACCAGGCTCCAAACAGAGCCCTCCAATGGCTTCCAG

[0096] CCCCATCCAGCCCAGGAAGGGAGGAGCTGACTGGTTGGGCCTCAAGGAC

[0097] GAGGACTTGGACCTGTTCCCTGCCTCACCCACCAGAGAGGCCCATCGGG

[0098] AAAGTTCAGTGCCTGTCACGCCCTCAGTGCCTCCTCCTGCGAGCCAGCAC

[0099] TCCACGCCAGCTGGGCTGCCCCCCTCCAGGGCAAAGCCACCAACTGAAG

[0100] GTGCAGGGTCCCCTGCCAAAGCCAGCCAGGCTTCCAAGCTGCGAGCCTC

[0101] CAAGGAGGAGAAAGAGGACTGGCTGAGCCATGCCCTGTCTCGGAAGAAG

[0102] TCCCAAGGCCTGGCCAGAGAGCAGCATGCTGGGACCTCTGAGGGCCTGC

[0103] ATTTGGCGGGACAGCGGGCCATCCCCCTTCTGGCAGCCAACCTCTCACC

[0104] AGCACACAAGGGCTTGAGCACGCAGCTGCTGGAGGGAGTTCTGGAACAA

[0105] CTGCACGAGAAAGACCGTGTGTCAGGCCTGGTGTCTCGGGGTCCCCTGT

[0106] GACTCAGAACCATGCCGCCTCAGCACTCCCTACAGGTTCCCCAAAGAGGG

[0107] GAACAGCCCCTGGAGACCTCTCAGCCACTGAGCCTGCCACGTGTTTCCCG

[0108] AGCACCCAGAAAACCCACAGAGCCTTCCGTGCCCGTCCAGCCCCTGCTCCC

[0109] AGAGTCCCTGGCCCGGAGCCTGCTGCCGAGCACAGAATACCAGAAGCAG

[0110] CTCCTGGCAGCACAGGTGCAACTTCAGTGCAGCCCGCTGAGCTCCAGG

[0111] CCGAGCTGCTGCATAGCCAGGCCCGGCTGGCAGAGCTGGAGGCCCAGGT

[0112] GCGGAAGCTGGAGCTAGAACGGGCCCAGCATGAGCTGCTGCTGGGGAGT

[0113] CTGCAGCAGCAGCACCAGGCAGACCTGGAGCTCATCGAGAGTGCACACA

[0114] GAAGCCGCATCAAGGTGCTAGAAACATCGTACCAGCAACGGGAGGAGCG

[0115] GCTCCGGAGAGAGAACGAAGAGCTGTCAGCTCGGTATCTGTCGCAGTGC

[0116] CAGGAGGCCGAACAGGCCCGTGCTGAGCTTACGGCCCAGCACCAGCGGC

[0117] GCTTGGCGGCCATAGCGCAGGAGAAGGACCAGGAAATGGAGCGGCTCCG

[0118] GGAGCTGCAGCGGGCGTCCATCCTAGACATGCGCAGAGACCACGAGGAG

[0119] CAGCTGCAGCGGCTAAAGCTGCTGAAGGACCGAGAGGTCGATGCGGCCA

[0120] CCAGTGCCACCTCCCACACGCGGTCCCTGAATAGCATCATCCACCAGATG

[0121] GAGAAGTTCTCCAGCAGCCTGCACGAGTTGTCCTCCCGCGTGGAGGCCTC

[0122] GCACCTCACCACCTCCCAGGAGCGGGAGCTGGGGATCCGGCAGCGTGAC

[0123] GAGCAGCTGCGGGCACTGCAGGAGCGGCTGGGCCAGCAGCAGCGGGAC

[0124] ATGGAGGAGGCGGAGCCGGCAACAGGAGGTCATCGGGAAGATGGAG

[0125] GCACGGCTGAATGAGCAGAGCCGGCTGCTGGAGCAGGAACGCTGGCGGGG

[0126] TGACTGCCGAGCAGTCCAAGGCGGAGTCCATGCAGCGCGCCCTAGAGGA

[0127] GCAAAGGAAGGTCACGGCCCAGCAGATGGCCATGGAAAGGGCGGAGCT

[0128] GGAACGGGCCAAGAGCGCCTTGCTGGAGGAGCAGAAGTCTGTCATGCTC

[0129] AAGTGCGGGGAGGAGCGGCGGCGCCTGGCTGCCGAGTGGGCGGAGTTCT

[0130] CCGCGCAGCAAAAGCTGAGTAAGGAGCGGCCGAGCGCGAGGCCGAGC

[0131] GGGCATTGCAGGTGGACACCCAGCGGGAGGGGCACCCTCATCAGCCTGGC

[0132] CAAGGAGCAGGCTGAGCTGAAGATCAGGGCCAGCGAGCTCCGGGCCGA

[0133] GGAGAAGCAGCTGGCAGCGGAGAGAGCAGCCCTGGAGCAGGAGCGGCA

[0134] GGAGCTGCGGCTGGAGAAGGAGAGGATCAACGCCACCGCCCTGCGTGTC

[0135] AAGCTCCGCGCCGAGGAGGTGGAGAGCATGAGCAAGGTGGCCTCCGAGA

[0136] AGTACGAGGAGGGGGAGCGGGCATTGCGCGAGGCCCAGCAGGTGCAGG

[0137] CAGAGCAGCAGGCCCGGTTGCAGGCGGTGCAGCAACAGCAGGAGCGGC

[0138] TGCGGAAGCAGGAGCAGCACATGCACCAGGAGCATCTGAGTCTGGCCCA

[0139] GCAGAGGCTGCAACTGGACCGCGCACGACAGGACCTGCCCTCTAGCCTC

[0140] GTGGGTCTGTTCCCCAGGGCCCAGGGCCCTGCAGCCTCCAGCCAGAGTG

[0141] CCCTCATGCCTCCTGCTCCCACCACCCGTTGGTGCAGCCAGCCGCCAACT

[0142] GGCCTGGACCCCAGCCCCTTGCACCTCCATGCCAGGCTGGCACTGCTGAG

[0143] GCACATGGCAGAGCAGGACCGTGACTTCTTGGAGAATGAACAGTTCTTCC

[0144] TGGAGACCCTGAAGAAAGGGTCCTACAATTTGACATCTCATTCAGCCTGASEQ ID NO.2:

[0145] mapktkkgckgsiddflgdllgddmtlpekpvklashtrdttgvsqmfpsskartksllgddvfstmagleeadaev

[0146] sgiseadpqallqamkdldgmdadilglkksnsapskkaakdpgkgelpnhpkpaggaiptkkslpspsssghqn

[0147] rrfssedledplrgllsydeggitkqppvtqsktasdkspstvrdqgpsipltpgdtpirkkeellfddgddimatlgfgd

[0148] spkaekrqigdqegprparstldellgrgmatkllarpgtgehrefkldkkyqrpqdsedmwgdedftfgayqptvv

[0149] ssegrqsrrqsvsrffadsgadpkgepgskqsppmasspiqprkggadwlglkdedldlfpasptreahressvpvt

[0150] psvpppasqhstpaglppsrakpptegagspakasqasklraskeekedwlshalsrkksqglareqhagtseglhla

[0151] gtaghppsgsqpltstqglehaaaggssgttarerpcvrpgvsgspvtqnhaasalptgspkrgtapgdlsatepatcfp

[0152] stqkptepsvpvqpllpeslarsllpsteyqkqllaaqvqlqcspaelqaellhsqarlaeleaqvrkleleraqhelllgsl

[0153] qqqhqadleliesahrsrikvletsyqqreerlrreneelsarylsqcqeaeqaraeltaqhqrrlaaiaqekdqemerlr

[0154] elqrasildmrrdheeqlqrlkllkdrevdaatsatshtrslnsiihqmekfssslhelssrveashlttsqerelgirqrde

[0155] qlralqerlgqqqrdmeeersrqqevigkmearlneqsrlleqerwrvtaeqskaesmqraleeqrkvtaqqmame

[0156] raeleraksalleeqksvmlkcgeerrrlaaewaefsaqqklskeraereaeralqvdtqregtlislakeqaelkirasel

[0157] raeekqlaaeraaleqerqelrlekerinatalrvklraeevesmskvasekyeegeralreaqqvqaeqqarlqavqq

[0158] qqerlrkqeqhmhqehlslaqqrlqldrarqdlpsslvglfpraqgpaassqsalmppapttrwcsqpptgldpsplh

[0159] lharlallrhmaeqdrdfleneqffletlkkgsynltshsa

[0160] The PCR reaction system is shown in Table 2. The reaction conditions are shown in Table 3.

[0161] Table 2 PCR reaction system

[0162]

[0163]

[0164] Table 3 Reaction conditions

[0165] temperature time 94℃ 5min 94℃ 30s Tm-5℃ 30s 72℃ 1min~3min go to 2 35cycles 72℃ 10min 4℃ forever

[0166] Then, agarose gel electrophoresis was performed, and the target band was recovered and purified, and then the recovered purified product and the vector pLV-EF1α-MCS-IRES-Bsd (abbreviated as pLV plasmid) were double-digested. The enzyme digestion reaction system is shown in Table 4 and Table 5. The reaction conditions are: 37°C for 2 hours.

[0167] Table 4 Recycling and purification product enzyme digestion reaction system

[0168] Components volume 10×NEB Buffer 5μl Nhe I 1μl XB 1μl PCR product recovery 30μl <![CDATA[ddH2O]]> 13μl Total 50μl

[0169] Table 5 Vector restriction enzyme digestion reaction system

[0170]

[0171]

[0172] Finally, the above-mentioned restriction PCR product was connected with the restriction pLV vector using T4 DNA ligase, and finally the FBF1 recombinant plasmid was constructed for overexpression of the FBF1 gene. The ligation reaction system is shown in Table 6.

[0173] Table 6 Ligation reaction system

[0174] Components Volume 10×Ligase Buffer 1μl DNA fragments 6μl pLV Plasmid 2μl T4 DNA ligase 1μl Total 10μl

[0175] 4. Construction of human breast cancer cell lines with stable overexpression of FBF1 gene and silencing of FBF1 gene

[0176] 4.1 Construction of human breast cancer cell line stably overexpressing FBF1 gene:

[0177] 4.1.1 Lentivirus packaging

[0178] Day 1: Resuscitate 293T cells and prepare plasmids.

[0179] Day 2: Cultivate cells. When 293T cells are fully grown, expand the culture by passage 1 to 2.

[0180] Day 3: Plate laying. (1) Aspirate the culture medium, wash with PBS, and add trypsin to digest the cells; (2) Add complete culture medium to terminate digestion, resuspend the cells, and centrifuge at 1000 rpm for 5 min; (3) Resuspend the cells, count them, and plate six-well plates, with 1×10 cells per well. 6 Cells, culture cells.

[0181] Day 4: Transfection. (1) Dilute the packaging plasmid to 500 ng / μl, and then mix them in a volume ratio of 1:1:1; (2) Add 3μl of the mixed packaging plasmid to the centrifuge tube; (3) Add 1.5μg of the target gene plasmid; (4) Add 250μl of opti-MEM, mix well, and let stand; (5) Take another 1.5ml centrifuge tube, add 250μl of opti-MEM and 7.5μl of Lipo2000, mix gently, let stand at room temperature for 5 minutes; (6) Mix the components in the two 1.5ml centrifuge tubes together, mix gently, and let stand at room temperature for 20 minutes; (7) Take the cells out of the incubator, slowly aspirate the culture medium, and then add 2ml of fresh culture medium; (8) Add the 500μl mixture that has been left to stand to the six-well plate and place it in the cell culture incubator for culture.

[0182] Day 5: Change medium: 16 hours after transfection, aspirate the medium, add 3 ml of fresh medium, and continue culturing.

[0183] Day 6: Collect viruses: After 48 hours, collect the viruses into 1.5 ml centrifuge tubes and store them in a -80°C refrigerator.

[0184] 4.1.2 Lentivirus infection of cells

[0185] Day 1: Digest and count the cultured cells and plate them in six-well plates, with 1-2×10 5 cells.

[0186] The next day: After the cell density reaches 30%, infection is performed.

[0187] (1) Aspirate the culture medium in the six-well plate and add 2 ml of culture medium without dual antibody, and add 3 ml to the WT group;

[0188] (2) Add 1 ml of virus to the corresponding well and mix well;

[0189] (3) Add 3 μl polybrene to each well;

[0190] (4) Centrifugation at room temperature, 1600 rpm, for 1 hour;

[0191] (5) Aspirate the culture medium, add 3 ml of new culture medium, and place in the incubator for culture.

[0192] 4.1.3 Screening

[0193] (1) 48 hours after virus infection, the culture medium in the six-well plate was aspirated, and 3 ml of medium containing BSD was added to each well to start screening; (2) When all the cells in the WT wells were killed, the screening was continued for 3 days to ensure that all uninfected cells were eliminated; (3) The screening was stopped, the medium was replaced with normal medium, and the culture was expanded to finally obtain a human breast cancer cell line (T47D-FBF1) with stable overexpression of FBF1 and a control cell line (T47D-Ctrl).

[0194] 4.2 Construction of human breast cancer cell line with FBF1 gene silencing:

[0195] (1) Annealing: The annealing reaction system was prepared as shown in Table 7. Reaction conditions: 95° C., incubation for 5 min.

[0196] Table 7 Annealing reaction system

[0197] Components volume 100μM single-strand oligo(SEQ ID NO.5) 10μl 10×annealing buffer 2μl RNase-free water 8μl Total 20μl

[0198] The shRNA sequence used to silence the FBF1 gene is shown in SEQ ID NO.5.

[0199] SEQ ID NO.5:

[0200] 5'-AAAAGGACGGCATGGATGCTGATATTTGGATCCAAATATCAGCATC CATGCCGTCC-3'.

[0201] (2) Dilution of annealing product: After annealing is completed, collect the annealing product and place it at room temperature for 10 min. Then take 1 μl of the annealing product, add 499 μl of sterile water to dilute it, and mix well to obtain the diluted annealing product.

[0202] (3) Take 50 μl of the diluted product and perform agarose gel electrophoresis to check whether the annealing is successful, and store the remaining annealing product at -20°C.

[0203] (4) Ligation: Ligate the pLV-H1-EF1α-puro vector with the diluted annealing product. The reaction system is as shown in Table 8. Reaction conditions: 4°C overnight.

[0204] Table 8 Reaction system

[0205] Components volume Diluted annealing product 6μl pLV-H1-EF1α-puro vector 2μl Ligation buffer (10×) 1μl T4 DNA Ligase 1μl Total 10μl

[0206] (5) After transformation and plasmid extraction, the pLV-H1-shFBF1-puro vector was successfully constructed. According to the above steps, the control sequence was used to construct the pLV-H1-shRNA-scramble-puro vector as a control group vector. The control sequence is shown in SEQ ID NO.6.

[0207] SEQ ID NO.6:

[0208] 5'-AAAAGCTACACTATCGAGCAATTTTGGATCCAAAATTGCTCGATAGTGTAGC-3'.

[0209] (6) Lentivirus was packaged according to the previously described method, and MDA-MB-231 cells were infected with lentivirus carrying pLV-H1-shFBF1-puro or pLV-H1-shRNA-scramble-puro, and then treated with puromycin to obtain a cell line with stable FBF1 silencing (MDA-MB-231-shFBF1) and a control cell line (MDA-MB-231-shCtrl).

[0210] 5. Real-time quantitative PCR (qRT-PCR)

[0211] SOX2, OCT4, KLF4 and NANOG are important transcription factors for maintaining the stemness of tumor cells. Moreover, these transcription factors can be used as marker molecules of tumor stem cells to characterize the stemness of tumor cells.

[0212] Total RNA was extracted from the corresponding cells using Trizol (Takara), and RNA was reverse transcribed into cDNA using EasyTaq DNA polymerase reverse transcription kit (TRNAS). SYBR Green Master Mix (Thermo Fisher Scientific) reagent was then used to complete qRT-PCR by Applied Biosystems QuantStudio3 Real-time PCR system (ThermoFisher Scientific). The expression of each gene was measured by ΔΔCt method with reference to the expression of internal reference gene GAPDH. The primers used in the present invention were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd., and the specific sequences are shown in Table 9.

[0213] Table 9 Primers used for qRT-PCR

[0214] Gene Forward primer Reverse primer MYLK3 SEQ ID NO.7: GGGCAAGACCTGCTTAACAAC SEQ ID NO.8: GCACATGCTCTGCAACTTCT NID1 SEQ ID NO.9: AATCCCTGCTACATCGGCAC SEQ ID NO.10: CCACACACTGAGGGTTGTTCT HAPLN1 SEQ ID NO.11:ATGCTGTGATCGCCTCCTTC SEQ ID NO.12: CTCTGGGCTTTGTGATGGGA AKT3 SEQ ID NO.13: CGCACACGTTTCTATGGTGC SEQ ID NO.14:ATGGTGGCTGCATCTGTGAT COL3A1 SEQ ID NO.15: CGCCCTCCTAATGGTCAAGG SEQ ID NO.16:TTCTGAGGACCAGTAGGGCA CTAGE6 SEQ ID NO.17: CCATTGGGTCAAGCGTTCATC SEQ ID NO.18: TCTTGGGCCTCTTCCTCCTC ACKR3 SEQ ID NO.19: CACGTCTGCGTCCAACAATG SEQ ID NO.20: GCTTCTCCTGGTCACTGGAC ACSL1 SEQ ID NO.21:ACTACTTGACAGCGACGAGC SEQ ID NO.22: TTGGTCTGGTTTCCGAGAGC SOX2 SEQ ID NO.23: GGATAAGTACACGCTGCCCG SEQ ID NO.24:ATGTGCGCGTAACTGTCCAT OCT4 SEQ ID NO.25: GCTCGAGAAGGATGTGGTCC SEQ ID NO.26:CGTTGTGCATAGTCGCTGCT FBF1 SEQ ID NO.27: AGGTTCTTCGCAGACAGTGG SEQ ID NO.28: CAGGGAACAGGTCCAAGTCC

[0215] 6. Western blot

[0216] (1) Extraction and concentration determination of total protein: Lyse the cells to be treated with RIPA buffer (Biyuntian) containing 1nmol / L PMSF (Biyuntian), extract total protein, and use BCA protein concentration determination kit (Biyuntian) to determine the concentration and quantify the different groups of proteins, then boil in a 100℃ metal bath for 10 minutes and store at -80℃ for later use. (2) Prepare polyacrylamide gel: Prepare 10% polyacrylamide gel according to the molecular weight of the protein. Clean the 1.5mm glass plate used for gel preparation, dry it, align it, put it into the clamp and clamp it, clamp it vertically on the rack, prepare the gel preparation reagent according to the instructions, add the separation gel first, add it carefully to avoid bubbles, let it stand for 30 minutes, then add the concentrated gel to the top, insert the comb, be careful not to generate bubbles, let it stand for 30 minutes, and wait for the gel to solidify. (3) Sample loading and electrophoresis: Add the prepared protein samples to the sample loading wells in a certain order. Each group of experimental samples is separated by a protein marker. The starting voltage is 80V. Run the gel for 40 minutes, then change the voltage to 110V and continue electrophoresis for about 90 minutes. When the small molecule proteins are pulled away, stop electrophoresis. (4) Transfer: Take a PVDF membrane of appropriate size and put it into anhydrous methanol for activation. Then cover the PVDF membrane on the gel after electrophoresis, assemble it together with the transfer clamp, and use a glass rod to drive away bubbles. Put it into the transfer tank, 100V, 100min. (5) Blocking: After transfer, take out the PVDF membrane, put it into the freshly prepared blocking solution, and block it at room temperature on a shaker for 1h. (6) Incubate the primary antibody: After blocking, add the corresponding diluted primary antibody to the antibody incubation box and incubate it at 4℃ overnight on a shaker. (7) Incubate with secondary antibody: Take out the PVDF membrane incubated overnight, wash it with TBST for 3 times, 10 min each time, then add the prepared secondary antibody, and incubate it on a shaker at room temperature for 1 hour. (8) Exposure and development: Wash the PVDF membrane with TBST for 3 times, 10 min each time, dry the washed membrane with absorbent paper to remove excess liquid, evenly add developer, and take pictures with an imaging system and save them. The primary antibodies used in the present invention are FBF1 (Proteintech, 11531-1-AP), SOX2 (CST, 3579), OCT4 (CST, 2840), KLF4 (CST, 4038), NANOG (CST, 4903), PI3K (CST, 4292), p-PI3K (CST, 4228), AKT (CST, 4691), p-AKT (CST, 4060), and actin (Santa Cruz, sc-47778).

[0217] 7. Side population experiment

[0218] (1) Digest and treat the cells, count them accurately, and take 1×10 6Place the cells into a flow cytometry tube, label it, centrifuge it, and resuspend the cells in 1 ml of PBS containing 2% FBS. (2) Add 5 μl Hoechst 33342 (final concentration 8 μg / ml) and 5 μM reserpine to the Block group, and add 5 μl Hoechst 33342 to the experimental group. (3) Incubate at 37°C in the dark for 1 hour, shaking every 10 minutes. (4) After staining, centrifuge at 1000 rpm, 4°C for 5 minutes, discard the supernatant, add 1 ml of pre-cooled PBS containing 2% FBS to resuspend the cells, and then add 1 μl of 1 mg / ml PI staining solution, and then detect by flow cytometry.

[0219] 8. Sphere formation

[0220] (1) Prepare spheroidization medium: basic medium (DMEM / L15) + 20ng / ml bFGF + 20ng / ml hEGF + 1×B27, 20ng / ml bFGF and 20ng / ml hEGF are both final concentrations. (2) Plating: Trypsinize the cells, count them accurately, and plate them on a non-adherent 48-well plate at 100-500 cells / 300μl spheroidization medium per well. Culture them in a cell culture incubator for 2 weeks. (3) After the cells are spheroidized, observe them under a microscope and count the number of cells in each group.

[0221] 9. Animal Experimentation

[0222] Female nude mice aged 6 to 8 weeks were selected, and animal experiments followed the guidelines of the "Ethics Committee of Xinxiang Medical College". After the cells used in the nude mouse tumor experiment were cultured to the logarithmic growth phase, the cells were collected and inoculated into the mammary fat pad of the mice. The tumor size was measured every 5 days, and the mice were processed after 45 days, the tumor weight was weighed and photographed, and the lung tissue of each mouse was taken and then embedded in paraffin for the next step of research.

[0223] 10. Transcriptome sequencing (RNA-seq)

[0224] T47D-Ctrl and T47D-FBF1 were used as research objects, with 3 replicates in each group. After the cells were cultured to the logarithmic growth phase, the cells were collected to extract RNA, the concentration and purity of RNA were determined, and then the samples were sent to BGI Sequencing Company for transcriptome sequencing. BGI was responsible for the subsequent library construction and transcriptome data analysis.

[0225] 11. H&E staining

[0226] (1) Dewax the paraffin sections, then rinse with 0.1% PBST 3 times, 5 minutes each time. (2) Wipe the tissue sections clean, add hematoxylin to cover the tissue, incubate for 1-3 minutes, then wash with tap water for 1-2 minutes. (3) Dye with eosin for 1-2 minutes, then wash with tap water for 1 minute. (4) Dehydrate the tissue sections, then seal with neutral gum and observe under a microscope.

[0227] 12. Statistical analysis

[0228] GraphPad Prism 5 and other software were used for statistical analysis. * p<0.05, ** p<0.01, *** p<0.001.

[0229] 2. Experimental Results

[0230] 1. Analysis of FBF1 expression in breast cancer and normal samples

[0231] The expression of FBF1 in breast cancer and normal samples was analyzed using the online analysis website UALCAN. The results showed that compared with normal tissue samples, the expression of FBF1 in breast cancer was significantly increased ( Figure 1 ).

[0232] 2. Effect of overexpression of FBF1 on the stemness of breast cancer cells

[0233] First, the effect of FBF1 on the proportion of side population cells in breast cancer was detected through a side population experiment. The results showed that overexpression of FBF1 can increase the proportion of side population cells in breast cancer T47D cells ( Figure 2 A and Figure 2 B).

[0234] The effect of FBF1 on the stemness of breast cancer cells was further explored through sphere formation experiments. The results showed that overexpression of FBF1 could enhance the sphere formation ability of breast cancer T47D cells. Figure 2 C and Figure 2 D), further indicating that FBF1 can promote the stemness of breast cancer cells.

[0235] The present invention further analyzed the effect of FBF1 on breast cancer stemness by qRT-PCR and Western blot. The results showed that overexpression of FBF1 can enhance the expression of SOX2, OCT4, KLF4 and NANOG ( Figure 2 E and Figure 2 F), which also indicates that FBF1 has the ability to promote the stemness characteristics of breast cancer cells.

[0236] 3. Effect of silencing FBF1 on the stemness of breast cancer cells

[0237] The present invention silenced the expression of FBF1 in MDA-MB-231 cells, and found that silencing FBF1 inhibited the expression of SOX2, OCT4 and NANOG ( Figure 3 A and Figure 3 B). At the same time, silencing FBF1 reduced the side population cell proportion and sphere-forming ability of MDA-MB-231 cells ( Figure 3 C. Figure 3 D. Figure 3 E and Figure 3 F).

[0238] 4. FBF1 can regulate the PI3K / AKT signaling pathway

[0239] In order to further analyze the molecular mechanism of FBF1 regulating breast cancer cell stemness, the present invention performed transcriptome sequencing (RNA-seq). The cluster heat map shows the main upregulated differentially expressed genes associated with tumor progression ( Figure 4 Next, in order to explore the key signaling pathways that FBF1 can affect, KEGG pathway classification showed that FBF1 was significantly associated with cell processes such as cell movement, cell growth and death ( Figure 4 B). At the same time, KEGG pathway analysis showed that these differentially expressed genes were mainly concentrated in signaling pathways such as PI3K-AKT and IL17 ( Figure 4 In addition, GO enrichment analysis showed that the differentially expressed genes were mainly concentrated in processes such as cell movement, cell proliferation, adhesion and growth ( Figure 4 D), and the analysis of molecular functions showed that it was mainly involved in the regulation of catalytic activity and other related functions ( Figure 4 E).

[0240] In order to further verify the accuracy of RNA-seq results, the present invention used qRT-PCR to detect the expression of these differentially regulated genes related to tumor progression. The results showed that compared with the control group, in T47D cells overexpressing FBF1, the expression of multiple genes such as AKT3 was significantly increased, which was consistent with the results of RNA-seq ( Figure 4 Combined with the results of qRT-PCR validation analysis and KEGG pathway analysis, the present invention further found through Western blot experiments that overexpression of FBF1 can enhance the expression of p-PI3K and p-AKT ( Figure 4 G). Taken together, the above results indicate that overexpression of FBF1 can activate the PI3K / AKT signaling pathway.

[0241] 5. PI3K inhibitor alleviates the effect of overexpression of FBF1 on the stemness of breast cancer cells

[0242] In order to gain a deeper understanding of the regulatory role of the PI3K / AKT signaling pathway in FBF1-mediated breast cancer cell stemness, the present invention added a PI3K inhibitor (LY294002) to T47D cells overexpressing FBF1. The results showed that overexpression of FBF1 increased the proportion of side population cells in breast cancer T47D cells, while the proportion of side population cells decreased significantly after the addition of the PI3K inhibitor ( Figure 5 A and Figure 5 At the same time, overexpression of FBF1 enhanced the spheroidization ability of T47D cells, but when PI3K inhibitor was added, the spheroidization ability was reduced ( Figure 5 C and Figure 5 The above results indicate that PI3K inhibitors can alleviate the promoting effect of overexpression of FBF1 on the stemness of breast cancer cells.

[0243] 6. FBF1 promotes breast cancer tumor growth and lung metastasis in mice

[0244] The present invention uses T47D cells that stably overexpress FBF1 and control group cells to conduct mouse tumor formation experiments. By detecting tumor volume and tumor weight, we found that overexpression of FBF1 can promote the growth of breast cancer tumors, while the use of PI3K inhibitors can slow down the volume and weight of tumors ( Figure 6 A~ Figure 6 C). At the same time, overexpression of FBF1 can promote lung metastasis of breast cancer, while the use of PI3K inhibitors reduces lung metastasis of breast cancer ( Figure 6 D~ Figure 6 F).

[0245] In summary, the present invention found that FBF1, as a new tumor stemness regulatory molecule, plays an important role in maintaining breast cancer stemness, providing a potential target for the diagnosis and precision treatment of breast cancer, which has important theoretical guidance significance and clinical application value.

[0246] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0247] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a product for detecting FBF1 gene or FBF1 protein in the preparation of a tool for detecting the stemness characteristics of breast cancer cells, characterized in that: The coding sequence of the FBF1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FBF1 protein is shown in SEQ ID NO.

2.

2. Use of the product for detecting FBF1 gene or FBF1 protein according to claim 1 in preparing a tool for detecting the stemness characteristics of breast cancer cells, characterized in that: The product for detecting the FBF1 gene or FBF1 protein includes a reagent for detecting the expression level of the FBF1 gene or FBF1 protein.

3. Use of the product for detecting FBF1 gene or FBF1 protein according to claim 2 in preparing a tool for detecting the stemness characteristics of breast cancer cells, characterized in that: The reagent for detecting the expression level of the FBF1 gene includes primers for amplifying the FBF1 gene shown in SEQ ID NO.3 to SEQ ID NO.

4.

4. Use of the product for detecting FBF1 gene or FBF1 protein according to claim 1 in preparing a tool for detecting the stemness characteristics of breast cancer cells, characterized in that: The means include nucleic acid capable of binding to FBF1 gene or a substance capable of binding to FBF1 protein.

5. Use of the product for detecting FBF1 gene or FBF1 protein according to claim 4 in preparing a tool for detecting the stemness characteristics of breast cancer cells, characterized in that: The substance capable of binding to the FBF1 protein includes an antibody that specifically binds to the FBF1 protein.

6. A shRNA that interferes with the FBF1 gene, characterized in that: The sequence of the shRNA is shown in SEQ ID NO.

5.

7. An interference vector containing the shRNA that interferes with the FBF1 gene according to claim 6, characterized in that: The shRNA interference vector is obtained by annealing the shRNA shown in SEQ ID NO.5 to generate a double-stranded product and then connecting it with the pLV-H1-EF1α-puro vector.

8. A lentivirus containing the interference vector according to claim 7, characterized in that: Obtained by packaging the shRNA interference vector with lentivirus.

9. Use of FBF1 gene inhibitor in the preparation of a drug for treating breast cancer, characterized in that: The FBF1 gene inhibitor has the shRNA that interferes with the FBF1 gene as claimed in claim 6 as the only effective ingredient.

10. Use of the FBF1 gene inhibitor according to claim 9 in the preparation of a drug for treating breast cancer, characterized in that: The drug has the following effects: Inhibit the stemness of breast cancer cells; Reduced the side population cell proportion and sphere-forming ability of breast cancer cells.

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