阅读说明：本技术 NSrp70基因在制备用于肿瘤转移相关药物制剂中的应用 (Application of NSrp70 gene in preparation of pharmaceutical preparation related to tumor metastasis ) 是由 金伟 赵阳 孙荷芬 于 2018-08-15 设计创作，主要内容包括：本发明属于生物医学工程技术领域,涉及NSrp70基因的新应用,特别NSrp70基因在制备用于肿瘤转移相关药物制剂中的应用。本发明对乳腺癌细胞进行了核蛋白定量蛋白组学分析,结果显示,核散斑蛋白NSrp70在具有高转移潜能的乳腺癌细胞呈现低表达,体内外实验证实干扰NSrp70能够显著促进细胞的转移能力,而过表达该基因则显著抑制细胞的转移能力；NSrp70能通过抑制TGFβ信号通路,抑制细胞EMT最终抑制乳腺癌转移；NSrp70可作为乳腺癌转移程中新的分子靶点以及分子标志物用于制备乳腺癌癌症转移相关药剂中的应用,包括制备检测癌症转移的检测试剂盒、抑制癌症转移的药物以及抑制与癌症转移相关信号通路的药物。本发明将有助于预防和抑制乳腺癌的转移,造福癌症患者。(The invention belongs to the technical field of biomedical engineering, and relates to a new application of NSrp70 gene, in particular to an application of NSrp70 gene in preparation of a medicine preparation related to tumor metastasis, wherein the result of the nuclear protein quantitative proteomics analysis on breast cancer cells shows that the nuclear speckle protein NSrp70 presents low expression in the breast cancer cells with high metastasis potential, in vitro and in vivo experiments prove that the interference NSrp70 can remarkably promote the metastasis capability of the cells, and the over-expression of the gene remarkably inhibits the metastasis capability of the cells, the NSrp70 can finally inhibit the breast cancer metastasis by inhibiting a TGF β signal channel, and the NSrp70 can be used as a new molecular target spot and a molecular marker in the breast cancer metastasis process for preparing a medicine related to the breast cancer metastasis, including preparation of a detection kit for detecting the cancer metastasis, a medicine for inhibiting the cancer metastasis and a medicine for inhibiting the signal channel related to the cancer metastasis.)

Use of NSrp70 gene or its fragment in the preparation of a pharmaceutical formulation for inhibiting breast cancer metastasis.

2. The use of claim 1, wherein the NSrp70 gene or fragment thereof is the major active ingredient of a pharmaceutical preparation for cancer metastasis from breast cancer.

3. The use of claim 1, wherein said cancer metastasis comprises metastasis, invasion and migration of cancer cellular components.

4. The use of claim 1, wherein said cancer metastasis suppressing agent comprises an agent that inhibits cancer metastasis, invasion, or migration.

Use of the NSrp70 gene or fragment thereof in the preparation of a pharmaceutical formulation for inhibiting signaling pathways associated with cancer metastasis.

6. The use according to claim 5, wherein the signaling pathway associated with cancer metastasis is the TGF- β signaling pathway.

Use of the NSrp70 gene or fragment thereof in the preparation of a test kit for the prognosis of breast cancer.

8. A kit for detecting cancer metastasis, comprising NSrp70 gene or a fragment thereof, or a polypeptide encoded by the gene or fragment.

9. A cancer metastasis detection kit, comprising an amplification primer specific to NSrp70 gene or an antibody specific to NSrp70 protein.

10. The kit for detecting cancer metastasis according to claim 8 or 9, wherein said kit further comprises a positive control and a negative control or instructions.

Technical Field

The invention belongs to the technical field of biomedical engineering, and relates to a new application of an NSrp70 gene, in particular to an application of an NSrp70 gene in preparation of a tumor metastasis related medicinal preparation.

Background

The data disclose that breast cancer has become a disease that seriously affects the physical and mental health of women worldwide, and the morbidity and mortality rate are rising year by year. Research shows that breast cancer metastasis is the main cause of breast tumor death, and clinical practice shows that breast cancer can transfer to a plurality of organs including lung, bone, liver, pleura, soft tissue and the like; wherein the median survival time of soft tissue and bone metastases is 22-26 months, lung and pleural metastases is 10-12 months, and liver and brain metastases are 4-6 months. Breast cancer metastasis rarely cures once it occurs, and the prognosis is poor, significantly reducing the quality of life of patients and increasing treatment costs.

Currently, the clinical treatments for metastatic breast cancer are classified into radiotherapy and chemotherapy and targeted therapy; compared with breast cancer of Luminal and HER2+, TNBC cannot adopt conventional endocrine therapy and targeted therapy due to the fact that ER and HER2 are not expressed, chemotherapy is limited by drug resistance, prognosis of patients is generally poor, and an effective treatment scheme is needed clinically.

From the above, there is still much room for improvement in the treatment of breast cancer metastasis. Therefore, the screening of the breast cancer metastasis marker is enhanced, the early diagnosis marker of the breast cancer metastasis and the potential target of prognosis are actively searched, and the breast cancer metastasis marker is applied to the development and application of related medicaments of breast cancer, so that breast cancer patients can be benefited, the life of the breast cancer patients is prolonged, and the life quality is improved.

Based on the current state of the prior art, the inventors of the present application propose to provide a new use of the NSrp70 gene, in particular, the use of the NSrp70 gene in preparing a pharmaceutical preparation related to tumor metastasis.

Disclosure of Invention

The invention aims to provide a new medicinal application of the NSrp70 gene based on the current state of the prior art, in particular to an application of the NSrp70 gene in preparing a medicinal preparation related to tumor, especially malignant tumor (also called cancer) metastasis, which comprises a detection kit for detecting cancer metastasis, a medicament for inhibiting cancer metastasis and a medicament for inhibiting a signal pathway related to cancer metastasis.

According to the invention, the iTRAQ quantitative proteomics is utilized to carry out nucleoprotein quantitative proteomics analysis on breast cancer cells MDA-MB-231 (parent cell line) and high-transfer cells MDA-MB-231HM (lung high transfer) and MDA-MB-231Bo (bone high transfer) derived from the breast cancer cells, and the result shows that the nuclear speckle protein NSrp70 presents low expression in the breast cancer cells with high transfer potential, and then, in vitro and in vivo experiments further prove that the interference NSrp70 can remarkably promote the transfer capability of the cells, and the over-expression of the gene remarkably inhibits the transfer capability of the cells, so that NSrp70 can inhibit the breast cancer transfer finally by inhibiting a TGF β signal channel, the breast cancer clinical specimen detection is carried out, the high expression of NSrp70 is shown to have better prognosis, and experimental researches show that NSrp70 can be used as a new molecular target in the breast cancer transfer process, and can be used as a molecular marker for clinical prevention, breast cancer, screening and screening of more patients with potential transfer risks in the early stage.

The invention provides a new application of an NSrp70 gene, namely an application of an NSrp70 gene or a fragment thereof in preparing a medicament related to breast cancer metastasis, which comprises the step of using the NSrp70 gene or the fragment thereof in preparing a detection reagent kit for detecting cancer metastasis, a medicament for inhibiting cancer metastasis and a medicament for inhibiting a signal path related to cancer metastasis.

The NSrp70 of the invention refers to a Gene for coding the nuclear speckle protein NSrp70 (also known as CCDC 55; HSPC095, NCBI website Gene ID: 84081).

In the present invention, the NSrp70 gene fragment refers to a nucleotide sequence encoding a polypeptide having the activity of human NSrp70 protein, such as nucleotide sequence at positions 38 to 1714 in the sequence having NCBI accession No. NM _032141 and degenerate sequences thereof; the degenerate sequence is a sequence of nucleotides 38-1714 of the coding sequence in which one or more codons have been replaced by degenerate codons encoding the same amino acid; due to the degeneracy of codons, degenerate sequences with homology as low as about 70% to nucleotide sequences 38-1714 can also encode the same polypeptide; also included are nucleotide sequences that hybridize under moderately stringent conditions, more preferably under highly stringent conditions, to a nucleotide sequence of the sequence selected from nucleotides 38 to 1714; also included are nucleotide sequences having at least 70%, preferably at least 80%, more preferably at least 90% homology with the sequence from nucleotide 38 to 1714.

The NSrp70 gene fragment also includes variants of the NSrp70 gene sequence that encode proteins with the same function as human NSrp70, including (but not limited to): deletion, insertion and/or substitution of several (usually 1 to 90, preferably 1 to 60, more preferably 1 to 20, most preferably 1 to 10) nucleotides, and addition of several nucleotides at the 5 'and/or 3' end; the coding sequence of the invention may be DNA or RNA, and may be single-stranded or double-stranded.

The NSrp70 gene or its fragment is the main active component of breast cancer metastasis agent.

The novel application of the NSrp70 gene comprises the application of the NSrp70 gene or the fragment thereof in preparing a detection kit for detecting cancer metastasis, a medicament for inhibiting cancer metastasis and a medicament for inhibiting a signal path related to cancer metastasis.

The cancer metastasis includes metastasis, invasion and migration of cancer cell components.

The cancer metastasis medicament comprises a medicament for inhibiting cancer metastasis, invasion and migration.

Such agents include drugs that inhibit signaling pathways associated with cancer metastasis, e.g., in a preferred embodiment of the invention, silencing NSrp70 activates the TGF- β signaling pathway and results in breast cancer or cancer metastasis thereof.

In the invention, the NSrp70 gene or the fragment thereof can be used for preparing a detection kit for breast cancer prognosis.

The NSrp70 gene or a fragment thereof can be used as an active ingredient of a cancer metastasis kit.

The invention provides a cancer metastasis kit, which comprises NSrp70 gene or fragment thereof, or polypeptide encoded by the gene or fragment.

The kit of the present invention may further comprise a primer specific for amplifying the NSrp70 gene or an antibody specific for the NSrp70 protein.

The kit of the invention also comprises a positive control and a negative control or instructions.

The NSrp70 gene and its expression product can be used as the main active component of the medicine for inhibiting cancer metastasis.

The proteins of the present invention, and antibodies, inhibitors, antagonists or receptors thereof, and the like, when administered (dosed) therapeutically, provide different effects; generally, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally from about 5 to about 8, preferably from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated; the formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking the human NSrp70 protein of the invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier; such pharmaceutical compositions comprise a therapeutically effective amount of the protein and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the administration mode. The human NSrp70 protein of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions, such as tablets and capsules, can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

When the human NSrp70 protein polypeptide of the invention is used as a medicament, a therapeutically effective dose of the polypeptide can be administered to a mammal. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

The invention provides application of NSrp70 in preparing a cancer metastasis related medicinal preparation, which comprises application in preparing a detection kit for detecting cancer metastasis, a medicament for inhibiting cancer metastasis and a medicament for inhibiting a signal path related to cancer metastasis, and the content and the expression condition of NSrp70 are detected by a series of methods and technologies. Further, the above-mentioned application contributes to the clinical early detection and poor prognosis of cancer metastasis.

Drawings

Fig. 1 is a mass spectrum result of nuclear speckle protein NSrp 70;

FIG. 2 is a graph showing the results of proteomics showing the differential expression of NSrp70 in MDA-MB-231, MDA-MB-231HM, MDA-MB-231Bo cell lines;

FIG. 3 is a graph showing the results of Western blot to verify the expression of NSrp70 in different cell lines of breast cancer;

FIG. 4 is a graph showing the results of Western blot to verify the effect of interfering with expression of NSrp70 in breast cancer cells;

FIG. 5 is a graph of the results of Western blot demonstrating the effect of over-expressing NSrp70 in breast cancer cells;

FIG. 6 is a graph of the results of Transwell validation of the effect on invasion and migration after perturbation of NSrp70 expression in MDA-MB-231, where the top is a representative picture and the bottom is a histogram of the experimental results;

fig. 7 is a graph of results of Transwell validation of the effect on invasion and migration after interfering with NSrp70 expression in BT549, with representative pictures of experimental results on the top and bar charts of experimental results on the bottom;

FIG. 8 is a graph of the results of Transwell's validation of the effect on invasion after overexpression of NSrp70 in Hs578T, where the upper part is a representative picture and the lower part is a histogram of experimental results;

FIG. 9 is a graph of the results of Transwell validation of the effect on invasion after overexpression of NSrp70 in MDA-MB-231HM, where the upper part is a representative picture of the experimental results and the lower part is a histogram of the experimental results;

FIG. 10 is a graph of the results of a scratch test to verify the effect on cell healing capacity following perturbation of NSrp70 expression in MDA-MB-231, wherein the upper part is a representative picture of the results of the test and the lower part is a linear statistical graph of the results of the test;

fig. 11 is a graph of the results of the scratch experiment to verify the effect on cell healing capacity following perturbation of NSrp70 expression in BT549, where the upper part is a representative picture of the experimental results and the lower part is a linear statistical graph of the experimental results;

fig. 12 is a graph of the results of mouse in vivo imaging. The upper part is the result of PCDS and the lower part is the correlation result of NSrp 70.

FIG. 13 shows the change of EMT-related index after detecting interference of NSrp70 with MDA-MB-231 by Western Blot.

FIG. 14 shows the change of EMT-related markers after Western Blot to detect over-expression of NSrp 70.

FIG. 15 shows the changes of EMT related indexes after detecting that NSrp70 interferes with BT549 and the changes of EMT related indexes after treating TGF β signal channel inhibitor GW788388 by Western Blot.

FIG. 16 is the result of in vitro migration of TGF β signaling pathway inhibitor GW788388 after treatment of NSrp70 interfering with MDA-MB-231.

Figure 17 correlation of detection of NSrp70 in clinical specimens with patient prognosis.

Fig. 18 is a graph showing the results of detecting NSrp70 and the lymph node metastasis status of a patient in clinical specimens.

Detailed Description

The embodiment of the invention comprises the following steps:

materials and methods

1 Material

1.1. Cell lines

The immortalized human mammary gland epithelial cell line MCF10A, the metastatic mammary gland cancer cell line MCF10-Ca1a from the same patient, and commonly used human mammary gland cancer cell lines MDA-MB-231, BT-549, Hs-578T, MCF-7, SK-BR-3, T47D, Bcap-37, MDA-MB-468, ZR-75-30 and ZR-75-1 were purchased from cell banks of the Chinese academy (Shanghai). The lung high metastatic potential cell line MDA-MB-231HM is established in the early stage of the experiment, is separated and identified after four times of mouse tail intravenous injections and is patented, and is used for issuing articles in the laboratory for many times. Bone height-transformed cells MDA-MB-231Bo was gifted by Dr.Toshiyuki Yoneda (The university of Texas, Houston). The lung hyper-transforming cell line MDA-MB-231LM2 was donated by Joan Massague (Memorial Sloan-Kettering Cancer Center).

1.2 sources, strains and breeds of nude mice

In vivo transfer experiment, 6-8 week old BALB/c (nu/nu) female nude mice are selected for experiment, and the nude mice are purchased and cultured in SPF level animal room of laboratory animal department of Shanghai Slek laboratory animal liability company Limited.

1.3 clinical data

All clinical specimens in this section were from cases collected in 2002 to 2006 in breast surgery of subsidiary tumor hospital of the university of Compound Dane, with informed consent from the samplers. Because different molecular types of breast cancer exist, the treatment and prognosis of the breast cancer with different types are different. Therefore, when selecting a sample for preparing a tissue chip, 250 invasive ductal carcinoma specimens including 50 Luminal A-type specimens, 50 Luminal B-type specimens, 50 Her 2-positive specimens, and 100 triple-negative breast carcinomas were randomly extracted from different types by first classifying the specimens according to molecular classification from a large number of cases. These cases all had complete pathological diagnostic data and follow-up records, with histological grades 1-3. The clinical data include age, menstruation status, histological grade, tumor size, lymph node status, ER, PR, HER2 status, recurrence or metastasis time, and overall survival time. The follow-up visit information expiration date is 8 months in 2013, and the median follow-up visit time is 98 months.

1.4 Primary reagents and consumables

1.4.1 cell culture-related reagents and consumables

DMEM and RPMI-l640 medium were purchased from Hyclone, DMEM/F12, Fetal Bovine Serum (FBS), horse serum, 0.25% trypsin, 0.05% trypsin and insulin from Gibco; penicillin streptomycin double antibody (source culture) cholera toxin and DMSO (dimethyl sulfoxide) were purchased from Sigma; EGF (cell epithelial growth factor) was purchased from Millipore Bio; 75cm culture flasks, 10cm and 6cm petri dishes, cell culture plates of various specifications, 3mL and 10mL sterile pipettes were purchased from Corning Inc.

1.4.2 protein extraction quantification and Western blot related reagent and consumable

Protein extraction and quantification:

cathepsin lysate T-PER (Pierce), 100 Xprotease inhibitor (APExBIO), 100 Xphosphatase inhibitor (APExBIO) and BCA protein quantification kit (Solyebao).

Reagents and equipment required for WB:

30% acrylamide (29% acrylamide: 1% N, N-methylenebisacrylamide) (Solaibao), sodium dodecyl sulfate (SDS, Sigma), Ammonium persulfate (Ammonium persulfate, Sigma), TEMED (Sigma) PVDF membrane (Millipore), primary anti-diluent (Bilun), ECL electrochemiluminescence substrate (Millipore), polysorbate (Tween-20, Millipore), NaCl, KCl, Na₂HPO₄、KH₂PO₄(Chinese medicine), skimmed milk (BD), 20 × DTT (Fermentas), 5 × loading buffer (Fermentas), PageRuler^TMPrestaining protein marker (Fermentas), transfer membrane with three layers of filter paper (Bio-Rad, USA), glass and comb (Bio-Rad, USA).

1.4.3 reagents and consumables for vector construction

pCDH vector pCDH-CMV-MCV-EF1-puro (SBI), Taq enzyme (Fermentas), dNTP (Takara), BamHI and EcoRI (NEB), DH5 α competent cell (Takara), plasmid minipump kit (Tiangen), general agarose gel DNA recovery kit (Tiangen), general DNA product purification kit (Tiangen), DL2000DNA marker (Takara), 1kb DNA gradient marker (Fermentas), T4DNA ligase (Fermentas), tryptone (oxyid, UK), yeast extract (oxyid, UK), NaCl, agar (Byunnan), ampicillin (Sigma), plasmid macropump kit (Tiangen);

NSrp70 gene CDS overexpression primer synthesis (jinzhi) with the following specific sequence:

Forward primer:5’-CCGgaattc GCCACC ATG GAC TAC AAG GAC GAT GAT GACAAG CTC GAT GGA GGA ATGGCGATTCCGGGCAGGCA-3’(SEQ ID NO 1)；Reverse primer:5’-CGCggatcc TCAATCATCTTCTTTCTCAA-3’(SEQ ID NO 2)

NSrp70 interference adopts a criprpr/cas 9 double plasmid system, the vector is gRNA, and NSrp70 knockout vector construction primers are as follows:

Crispr#1:Forward primer:5’-CACCGAATGCAGCTTTCGTGAAGCC-3’(SEQ ID NO 3)

Reverse primer:5’-AAACGGCTTCACGAAAGCTGCATTC-3’(SEQ ID NO 4)

Crispr#2:Forward primer:5’-CACCGTAAGAAGCAGGCCATGAAAC-3’(SEQ ID NO 5)

Reverse primer:5’-AAACTAAGAAGCAGGCCATGAAACC-3’(SEQ ID NO 6)

1.4.4 viral packaging and reagents required for infection

Linearized Polyethyleneimine (PEI) (Polyscience), 0.9% NaCl (Chinese medicine), Polybrene (dimethylammonium bromide, Sigma), PsPAX₂、pMD₂G. Plasmid of interest (laboratory-stored plasmid), syringe (Shanghai gold tower), filter (Millipore), cell culture flask (Corning), EP tube (Invitrogen), and cell culture medium (Corning).

1.4.5 reagent consumables for cell function test

0.8 μm 24-well transwell chamber (BD); matrigel prefabricated 24-well transwell chamber (BD);

scratch specific 96-well plates (Essen).

1.4.6 reagent consumables for immunohistochemistry

NSrp70 antibody (Sigma), primary-anti-secondary antibody dilution buffer (Thermo fisher), mouse/rabbit universal immunohistochemical detection kit (DAB color, REAL EnVision), EDTA buffer: 50 × EDTA (pH 9.0, DAKO), 0.01M citrate buffer (Solarbio), hematoxylin (Solebao), a water-proof pen for immunohistochemistry (GT PenMini), Tips heads of different specifications (Axygen), a cover glass, a washing bottle (Fujian Mixin), a hatching wet box (Leica), 24 plastic staining rack, and a high temperature resistant antigen retrieval box (Shanghai Kagaku laboratory Co., Ltd.).

1.5 Main Instrument

Biological safety cabinets (Thermo Fisher);

37℃CO₂a constant temperature incubator (Thermo Fisher);

light microscope BX51 (Olympus);

vertical electrophoresis tank (Bio-Rad corporation);

an electrophoretic transfer chamber (Bio-Rad);

a Mettler PE-160 type electronic balance (Mettler);

low temperature high speed centrifuge (Eppendoff);

a normal temperature Centrifuge 5810R (Eppendorf);

MultiSKAN MK3 microplate reader (Thermo);

ice maker (SIM-F140, SANYO);

a pH meter (Mettler-Toledo);

vortex oscillator (IKA-MS);

a constant temperature water bath (Shanghai Biotechnology engineering Co., Ltd.);

ultra-pure water purification machines (Milli-Q);

a micropipette gun (Eppendorf);

shaker SCS-24 (Shanghai institute for centrifugal mechanics);

-20 ℃ refrigerator (Haier);

-80 ℃ refrigerator (Thermo Revco Scientific);

electronic balance (Mettler-Toledo).

High pressure steam cooker (san shen);

DNA electrophoresis apparatus and horizontal electrophoresis tank (Bio-Rad);

DNA gel imager (Tanon);

gel imager (WB) (Bio-Rad);

incucyte living cell workstation (Essen);

cell counter (Beckman);

small animal live imagers (Bruker);

conventional microtomes (Leica);

tissue chip instruments (Beecher instrument);

an electric heating constant temperature air-blast drying oven (Shanghai sperm macro experimental facilities Co., Ltd.).

1.6 preparation of culture Medium and conventional solutions

Cell culture related:

adding double antibody, 10% FBS and 4-degree refrigerator into DMEM, RPMI-L640 and L15 culture medium before use; MCF10A medium: DMEM/F12, 5% horse serum, cholera toxin, insulin (100X), hydrocortisone, epidermal growth factor (100ng/mL), diabody (100X).

Cell cryopreservation solution, DMSO: FBS: the culture medium is mixed uniformly according to the volume ratio of 1:2:7, and is used as the culture medium at present or stored in a refrigerator at 4 ℃ for avoiding light for storage.

10 × PBS buffer: 80g of NaCl, 2g of KCl and Na₂HO₄14.4g、KH₂PO₄2.4g。

PBST: 10 x PBS with double distilled water diluted to 1 x working concentration, added 0.1% concentration of Tween-20 and fully shake.

Preparation of Western blotting-related reagents:

Protein loading buffer Pack(5×Loading buffer，20×Reducing agent，Fermentas)；

running buffer (25mM Tris-HCl, 250mM glycine, 0.1% SDS);

1.0M Tris-HCl (pH6.8); 1.5M Tris-HCl (pH8.8); 10% (W/V) ammonium persulfate;

sealing liquid: 5% skim milk powder or 5% Bovine Serum Albumin (BSA) in 0.1M PBS (pH7.4);

20 times membrane transferring buffer solution, diluting to 1 times with deionized water when in use, and adding 20% methanol;

the acrylamide gel formula comprises:

preparing an LB culture medium:

sterilizing liquid LB under high pressure, and storing at 4 deg.C; sterilizing solid LB under high pressure, cooling to 50-60 deg.C (preferably not burning hands and back), adding appropriate antibiotic (such as ampicillin 100mg/mL), mixing, pouring into culture dish (about 10-15 mL), cooling, sealing, and storing in 4 deg.C refrigerator.

Crystal violet formula for Transwell staining: 0.445g Crystal Violet

71.2mL of methanol

106.8mL PBS

Filtering with three layers of filter paper, and storing in dark at normal temperature.

The formulation of a mouse living body imaging substrate D-luciferin potassium salt:

20mg/mL is dissolved in DPBS, filtered by a 0.22 mu m filter membrane and stored in the dark, and the compound is prepared for use.

(5) Formulation of DPBS:

reagent	Quality (g)	Molarity (mM)	Volume (L)
				NaCl	8	137	-
KCl	20	2.7	-
				Na2HPO4·7H2O	2.16	8.1	-
KH2PO4	0.20	1.1	-
				H2O	-	-	1

2 method

2.1 culture of cell lines

MCF10A uses its proprietary formulation; other breast cancer cell lines use the corresponding media according to ATCC recommendations. Culturing at 37 deg.C and 5% CO₂And saturated humidity conditions.

2.2 protein extraction

(1) Adherent cells: discarding the culture solution, washing the cells twice with precooled PBS and blotting, placing a cell culture dish or bottle on ice, adding a proper amount of protein lysate (containing protease inhibitor and phosphatase inhibitor), and scraping the cells with a cell scraper; cell pellet collected after trypsinization: washing twice with precooled PBS, discarding the supernatant, and directly adding lysis solution;

(2) collecting cell lysate into 1.5mL EP tube, using ultrasonic instrument to assist in breaking cell membrane (10s ultrasonic, 10s stop, total 4 cycles), and then cracking in ice bath at 4 deg.C for 40min, wherein multiple times of oscillation can be performed to ensure sufficient cell lysis;

(3) after centrifugation at 12000rpm at 4 ℃ for 15min, the supernatant was transferred to a new 1.5mL EP tube and the precipitate was discarded;

(4) mu.L was taken for determination of protein concentration, protein concentration determination (BCA method): 20 μ L PBS +5 μ L sample/5 μ L LPBS (blank control) +200 μ L BCA mixed solution (a: B ═ 200:4), water bath at 37 ℃ for 30min, OD value of a562 measured by microplate reader, and protein concentration was calculated according to standard curve;

(5) the remaining protein supernatant was denatured by adding 5 × loading buffer and 20 × DTT in boiling water bath for 10 min.

2.3Western blotting

(1) Preparing gel: respectively preparing polyacrylamide gel separation gel with different concentrations and 5 percent concentrated gel;

(2) protein loading: diluting the denatured protein sample to the same concentration with 1 × loading buffer, loading 30 μ g, protein marker 5 μ L;

(3) electrophoresis: performing 70V electrophoresis for 30min, performing 120V electrophoresis for 1-1.5h after the bromophenol blue enters the concentrated gel, and stopping electrophoresis when the bromophenol blue is about to leave the bottom end of the gel;

(4) film transfer: after electrophoresis is finished, transferring the protein to a PVDF membrane by adopting a wet transfer method, and carrying out membrane transfer conditions: constant current of 220mA for 50-90min, and properly adjusting according to molecular weight and gel thickness;

(5) and (3) sealing: PVDF membranes were rinsed once in PBST (PBS pH7.4, 0.1% Tween 20), immersed in 5% skim milk (or 5% BSA) blocking solution, and incubated slowly on a horizontal shaker at room temperature for 1 h;

(6) primary antibody incubation: diluting the primary antibody to a working concentration by using a primary antibody diluent, slowly shaking the antibody incubation box in a shaking table, and incubating the membrane at 4 ℃ overnight;

(7) and (3) secondary antibody incubation: after the primary antibody incubation is finished, washing the membrane for 4 times by PBST, and 8min for each time; diluting the secondary antibody with skimmed milk (or BSA), and incubating at room temperature for 1-2 h;

(8) substrate color development: after the secondary antibody incubation is finished, washing the membrane for 4 times by PBST, and each time for 8 min; and (3) taking the developing solutions A and B with the same volume, uniformly mixing, adding the mixture to the front surface of the PVDF membrane, and developing and taking a picture in an imaging analysis system.

All western blot experiments were performed with GAPDH as loading internal control.

2.4 recombinant wild-type NSrp70 expression vector construction

And (3) taking cDNA of the breast cancer cell line MDA-MB-231 as a template, obtaining a complete CDS sequence by utilizing PCR amplification, connecting the CDS sequence with a vector after enzyme cutting, and selecting monoclonal sequencing for verification. The method comprises the following specific steps:

2.4.1PCR amplification of the full Length of NSrp70CDS

The method comprises the following steps: firstly, selecting a restriction enzyme site according to a multiple cloning site on a vector, designing a primer required for amplifying an NSrp70CDS region, and diluting the primer to a working concentration of 10 mu M by using deionized water; secondly, diluting the template which is reversely transcribed into cDNA according to the ratio of 1:10 for later use; configuring a reaction system according to the following reaction, determining an annealing temperature range according to Tm values of upstream and downstream primers, carrying out annealing temperature gradient PCR, searching for the optimal annealing temperature, expanding the system to 200 mu L (50 mu L multiplied by 4 tubes), and carrying out gel purification to obtain fragments after the PCR is finished.

The PCR reaction system is as follows:

the PCR reaction conditions were as follows:

2.4.2 agarose gel purification of PCR products

(1) Preparing agarose gel: the size of the NSrp70CDS region is about 1677bp, and 1% agarose gel is prepared;

(2) adding a sample: the PCR product is mixed with 6 × loading buffer, carefully added into the sample loading hole, and 5 μ LDNA ladder 2000 is added;

(5) electrophoresis, namely performing electrophoresis at a constant voltage of 100V, and stopping electrophoresis when the bromophenol blue indicator is electrophoresed to about two thirds of the gel;

(6) photographing or cutting glue: after photographing and storing in a DNA imager, carefully cutting off a target strip by using a surgical blade, and repairing surrounding gel as much as possible;

(7) sol: the gel was cut into small pieces, filled into EP tubes and weighed. Adding sol solution (0.1g gel and 100 μ L sol solution), and dissolving in 50 deg.C water bath for 5-10 min. The last and last times of the mild reaction are repeated to ensure that all the gel is dissolved;

(8) column balancing: putting the adsorption column into a collecting tube, adding 500 μ L of equilibrium liquid, centrifuging at 12,000rpm at normal temperature for 30s, and discarding the waste liquid;

(9) adsorbing a target fragment: adding the uniformly mixed reaction solution obtained in the step 7) into the adsorption column obtained in the step 8), standing at room temperature for 2min, centrifuging at 12,000rpm for 1min, discarding waste liquid, and adding all samples in batch centrifugation;

(10) washing: adding 600 μ L of washing solution, standing at room temperature for 3-5min, centrifuging at 12,000rpm for 1min, and discarding the waste solution. Repeating the steps once;

(11) removing the washing liquid: centrifuging the adsorption column at 12,000rpm for 3min to remove the washing solution to the maximum;

(12) and (3) elution: putting the adsorption column into a new EP tube, opening the cover, standing at room temperature for 4-5min to ensure complete volatilization of alcohol. Then 50. mu.L of eluent is added into the column, the mixture is stood for 2min at room temperature and then centrifuged at 12,000rpm for 3min, and the liquid in an EP tube is collected, and the concentration of the liquid is measured by a Nanodrop 2000 and stored at-20 for later use.

2.4.3 destination fragment and pCDH vector double digestion

Enzyme cutting conditions are as follows: water bath at 37 deg.C overnight

The system is as follows:

2.4.4 purification of the cleavage products

And (3) cutting the vector DNA fragment, taking the large fragment after enzyme digestion, and purifying, wherein the experimental steps refer to 2.2.2.

And purifying the enzyme digestion product of the target fragment by using a common PCR product purification kit.

The method comprises the following specific steps:

(1) column balancing: putting the adsorption column into a collecting tube, adding 500 μ L of equilibrium liquid, centrifuging at 12,000rpm at normal temperature for 30s, and discarding the waste liquid;

(2) mixing the sample and the binding solution uniformly: adding 5 times of volume of binding solution into the PCR reaction solution, and fully and uniformly mixing;

(3) DNA adsorption: adding the uniformly mixed reaction solution into the adsorption column in the step 1), standing at room temperature for 2min, centrifuging at 12,000rpm for 1min, discarding waste liquid, and adding all samples by batch centrifugation;

(4) washing-removing washing liquid-eluting-measuring concentration: same as steps (10) - (12) in 2.3.2.

2.4.5 connection of target fragment and vector for recombination cloning

50ng of the vector was taken, and the mass of the fragment was calculated based on the molar ratio (target fragment: vector fragment: 7:1) and then a reaction solution was prepared as follows:

reaction conditions are as follows: PCR instrument at 22 deg.C for 60min

2.4.6 ligation product conversion

(1) Inserting the ligation product and DH5 α competent cells into ice, adding all the ligation products into the competence after the competence melts after about 4-6min, uniformly mixing, and standing on ice for 30 min;

(2) heat shock competence in 42 deg.C water bath for 90s, immediately placing on ice for 2-3 min;

(3) adding 900 μ L LB culture medium (without antibiotic), mixing, and shake culturing at 37 deg.C for 45-60 min;

(4) centrifuging at 4000rpm at room temperature for 2min, reserving about 100 μ L of bacterial liquid for resuspension, uniformly coating the bacterial liquid on an LB plate containing ammonia benzyl resistance, and inversely placing the bacterial liquid in a bacterial incubator at 37 ℃ for culture overnight.

2.4.7 testing and identifying by picking single clone

The next day, when the colony size is suitable, picking the monoclonal colony in an ultra clean bench, placing the colony in an LB shaking tube filled with 5mL ampicillin, marking, placing the colony on a 37 ℃ shaking table, and culturing overnight at 220 rpm.

Extracting recombinant plasmids by using a plasmid miniprep kit, performing agarose gel electrophoresis after double digestion, sequencing plasmids with correct target fragment sizes after digestion, and comparing the results by using Vector NTI (base 100% matching or mutation points are synonymous mutations).

The plasmid miniextraction procedure was as follows:

(1) and (3) bacteria collection: centrifuging overnight-cultured bacterial liquid at room temperature and 4000rpm for 10min, and completely sucking supernatant;

(2) and (3) resuspending the bacteria: adding 250 μ L of P1 liquid to each sample, and fully suspending the bacteria with a vortex oscillator;

(3) bacterial lysis: adding 250 μ L of solution P2, slowly reversing and mixing;

(4) protein precipitation and the like: adding 350 μ L of P3 solution, immediately reversing the temperature, mixing evenly, and centrifuging at 12000rpm for 15 min;

(5) column balancing: putting the adsorption column into a collection tube, adding 500 μ L of equilibrium liquid, centrifuging at 12000rpm at room temperature for 1min, and discarding the waste liquid;

(6) plasmid adsorption: carefully sucking out the supernatant obtained in the step 5), adding the supernatant into the adsorption column obtained in the step 6), standing for 2min, centrifuging at 12000rpm at room temperature for 2min, and removing waste liquid;

(7) washing-removing washing liquid-eluting: the same procedure as in 10) -12) of 2.3.2), the volume of eluent can be 80-100. mu.L.

2.5NSrp70 knock-out fragment vector construction

2.5.1 vector cleavage and recovery

The endonuclease is BsmbI, and after cutting, a fragment with the size of 8kd is selected for recovery, specifically seen in 2.4.3 vector enzyme cutting and 2.4.4 purified enzyme cutting products

2.5.2 primer annealing

Conditions are as follows: 4min at 95 ℃; 10min at 70 ℃; and (5) turning off the fire in the PCR.

The rest steps are the same as the construction of the over-expression vector.

2.6 packaging of viral and cellular infections

(1) Inoculating cells: culturing 293T to the density of about 80-90%, inoculating cells into a culture dish with the thickness of 60mm according to the ratio of 1:9, and standing overnight until the cells adhere to the wall, wherein the density after the cells adhere to the wall is about 70-80%;

(2) preparing a mixed solution of plasmid and a transfection reagent: the target plasmid: 4 mu g of the solution; packaging plasmid (psPAX)₂：3μg；

pMD₂G: 1.2. mu.g); 200. mu.l of 0.9% NaCl; 24 mu L of PEI as a transfection reagent; mixing all the reagents uniformly, shaking for 8s, and standing at room temperature for 10 min;

(3) transfection of cells: replacing 3mL of fresh non-double-antibody FBS preparation solution for 293T cells, slowly dripping the liquid in the step 2) into the culture solution, shaking up gently and crosswise, and putting into a cell culture box;

(4) liquid changing: after 6-8h, the cells were replaced with fresh medium (containing double antibody, 10% FBS);

(5) collecting virus supernatant: supernatants were collected 48h after transfection, filtered through 0.45 μm filters, and 1mL aliquots were dispensed into sterile EP tubes, directly infected cells or stored in a freezer at-80 ℃.

(6) Adding the virus solution and the fresh culture solution into cells with the cell density of 50-60% according to the ratio of 1: 1-1: 3, adding Polybrane with the final concentration of 8ug/ml, and replacing the fresh culture solution after the night. Stable transfected cell lines were obtained after selection of antibiotics for about 1 week based on vector antibody selection.

2.7 cell migration, invasion and scratch detection experiments

2.7.1 cellular wound healing Capacity test

(1) Inoculating cells: after cell counting at about 3.6X 10⁴Cells/well/100 μ Ι _ were seeded into Essen 96-well plates (about 90% confluence), adherent overnight;

(2) scratching: on the next day, filling blank holes in the 96-hole plate with 100-microliter PBS, then uncovering the 96-hole plate, placing the 96-hole plate in a scratching device for fixing, and scratching the 96-hole plate after disinfecting the 96-hole plate with 75% ethanol;

(3) removal of exfoliated cells: washing twice with 200 μ L PBS, removing the cells exfoliated after scratching, and adding 200 μ L fresh culture solution into each well;

(4) scanning: placing the 96-well plate in an Incucyte viable cell workstation, and scanning every 4 hours for 60 hours;

(5) and (3) analysis results: after the scanning is finished, the application software program analyzes the result, maps by taking the relative scratch density (relative power density) as a parameter, and derives the representative pictures of 0, 24 and 48 h.

2.7.2 cell migration and invasion (Transwell Chamber method)

(1) Cell counting: digesting the cells, resuspending the cells in complete medium, washing twice with DMEM without FBS, resuspending the cells, counting the cells, and adjusting the cell concentration to 2.5X 10⁵Perml (migration) and 5X 10⁵mL (invasion);

(2) cell inoculation: adding 600 mu L of DMEM medium containing 20% FBS into a 24-hole culture plate, placing a Transwell chamber (without prefabricated glue and with prefabricated glue) into a hole in which a culture solution is added in advance after ensuring that no air bubbles exist, uniformly blowing the cell suspension in the step (1), then dropwise adding 200 mu L of the cell suspension into the Transwell chamber, and placing the cell suspension into a cell culture box for culturing for 15-24 hours;

(3) cell staining: taking out the small chamber after the cells are cultured for a specific time, putting the small chamber into crystal violet staining solution for fixed staining for 30min, washing redundant staining solution with distilled water, wiping the cells in the small chamber clean with a cotton swab, and then inverting and airing;

(4) taking a picture and counting: and 4-time and 20-time objective lenses are selected under an inverted microscope to take pictures, at least 3 fields of each sample are taken and counted, and statistical analysis is carried out.

2.8 animal experiments

Packaging lentivirus by using PWPXL-GFP-luciferase plasmid, infecting required target cells, sorting GFP positive cells by a flow cytometer, then carrying out virus infection of target genes, and screening by puromycin and verifying interference and over-expression effects by WB.

Cell digestion and counting were resuspended in FBS-free DMEM at 3X 10⁶cell/mL; 3X 10 Vaccination from the caudal vein of mice⁵Cell/100 μ L, mouse body weight was recorded every three days; approximately 4-6 weeks or so, mice were anesthetized with ether and observed for metastasis in vivo with a Living imager after tail vein injection of 100 μ L D-luciferin potassium salt (20 mg/mL).

2.9 immunohistochemical staining

(1) Dewaxing and hydrating: taking out the chip from the refrigerator in advance, and baking the chip in an incubator at 60 ℃ overnight after the chip is returned to room temperature; the next day, the baked chips were placed on a staining rack and sequentially immersed in xylene I, ii, iii for 10min each (three times in xylene); taking out the chip, sequentially placing in 100% anhydrous alcohol I, II (twice anhydrous alcohol), 95% alcohol, 85% alcohol, and 75% alcohol, respectively standing for 5 min; subsequently removed and rinsed three times with PBS for 3min each. (dewaxing and hydration were carried out in a fume hood)

(2) Antigen retrieval: placing the antigen retrieval box filled with the citric acid buffer solution into an autoclave, turning on a power supply, and deflating and opening the cover after water in the autoclave is boiled; and (3) placing the staining rack with the chips in place into a pre-boiled antigen repairing box, continuously boiling for 10min, turning off a power supply, preserving heat for 5min, taking out the whole repairing box, and naturally cooling to room temperature. The cooled chips were rinsed with PBS for 5min and repeated three times.

(3) Non-specific blocking: taking out the chip, lightly throwing to remove excessive water, wiping off residual liquid around the tissue, drawing a circle around the tissue by using a water-proof pen, slowly dripping a proper amount of non-specific blocking agent on the tissue, and incubating for 10min at room temperature in a dark place. Followed by 3 washes with PBS for 5min each. The sections were removed, gently flung off excess fluid and carefully swabbing off the fluid surrounding the tissue to avoid over-drying the tissue.

(4) Primary antibody incubation: the chip was placed in a wet incubation box, 600. mu.L of diluted primary antibody working solution was gently added dropwise to the tissue, and incubated overnight at 4 ℃. The next day, the chip was placed in a 37 ℃ incubator and allowed to rewarming for 45 min. Subsequently, the chip was rinsed 3 times with PBS for 5min each, and the chip was removed to remove the liquid surrounding the tissue (the tissue was not overly dry).

(5) And (3) secondary antibody incubation: adding 600 μ L of secondary antibody working solution dropwise onto the tissue, incubating at 37 deg.C for 30min, placing the chip into PBS, washing for 3 times, each for 5min, taking out the chip, wiping off the excess liquid around the tissue, and keeping the tissue moist.

(6) DAB color development: mixing the freshly prepared color developing solution and a substrate according to a ratio of 1:50, then dripping 600 mu L of the mixture on a tissue, incubating at room temperature and observing under a microscope, controlling the color developing time for 1-5min, and after the color development is completed, flushing with tap water to stop the color development.

(7) And (3) returning hematoxylin to blue: washing the DAB developed chip with tap water for 5min, slightly wiping off the excess liquid around the tissue, adding 600 μ L hematoxylin, dyeing at room temperature for 2min, washing with tap water for 10min, adjusting water flow rate, and washing the back surface of the chip.

(8) Dehydration and transparency: the chips are respectively placed in alcohol with different concentrations (75%, 85%, 95%, 100%), each concentration gradient is 5min, and finally placed in dimethylbenzene I, II, III for 5min respectively. (dehydration and clarification in a fume hood)

(9) Sealing: the chip is dehydrated, transparent treated, sealed with normal neutral resin, and dried in a fume hood.

(10) Judging standard of immunohistochemical staining grade: the NSrp70 scoring criteria depend on the intensity and distribution of positive cell staining. The staining intensity was graded as follows: no positive staining was 0min, light yellow 1min, tan 2min, tan 3 min. The proportion of dyeing is graded from 0-4: the dyeing proportion is that 5 percent is 0, 5 to 25 percent is 1, 25 to 50 percent is 2, 50 to 75 percent is 3, and 75 percent is 4. The result of multiplying the dyeing ratio and the dyeing intensity is: low expression is rated at 0-3, and high expression is rated at 4-12. On the tissue chip, each case is repeated for two times, and the average value of the two is taken as the final score; if one of the repeats is missing, the score of the repeat hole is taken to replace the total score; if both repeats are missing on the chip, the case does not take the last statistical data into account.

(11)3 statistical treatment

The data of the part are statistically analyzed by adopting SPSS22.0 and chi²The test compares the differences between groups of clinically relevant parameters. Adopting Kaplan-Meier to analyze survival curve, adopting Cox analysis test to make single-factor and multi-factor analysis, adopting Spearman correlation analysis to make correlation between groups of counting data and using p<0.05 is that there is a statistical difference.