CN114703287B - Application of CXorf56 gene in treating triple negative breast cancer - Google Patents
Application of CXorf56 gene in treating triple negative breast cancer Download PDFInfo
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Abstract
The invention discloses application of CXorf56 gene or protein in preparation of a medicament for treating triple negative breast cancer or a synergist of the triple negative breast cancer medicament. The invention also discloses application of the recombinant vector, the recombinant cell and the recombinant protein for over-expressing the CXorf56 gene or knocking down the CXorf56 gene in preparing a medicament for treating triple negative breast cancer or a synergist thereof. The invention discovers that the CXorf56 gene can be knocked down to enhance the effect of the breast cancer medicament in treating the triple-negative breast cancer for the first time. Meanwhile, the fact that the reduction of the expression of the CXorf56 gene and the increase of the expression of the KU70 gene can further enhance the sensitivity of the breast cancer medicament in the treatment of triple-negative breast cancer, so that the treatment effect is improved.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to an application of CXorf56 gene in treating triple negative breast cancer.
Background
Breast cancer has leap forward to the first of female cancers worldwide, and the number of new cases of cancer reaches 230 ten thousand every year, accounting for 11.7 percent of all new cases of cancer. According to the immunohistochemical result, the breast cancer is roughly divided into four subtypes clinically, wherein Triple Negative Breast Cancer (TNBC) accounts for about 15%, the subtype has high invasiveness, early visceral metastasis generation and rapid development, and the life cycle of a patient is short. TNBC does not benefit from endocrine and anti-HER 2 therapy due to lack of female and progestogen receptors and HER2 expression, and has limited available therapeutic options and the worst prognosis. In recent years, more and more researches show that the BRCA1 gene is an important molecular target in TNBC treatment, and PARP inhibitors bring breakthrough progress to the treatment of BRCA1 mutant TNBC. However, in TNBC, only 10-20% of patients carry the BRCA1 mutation, and the low mutation rate leads to limitations in the use of PARP inhibitors; even if the BRCA1 mutation exists, the drug resistance ratio of TNBC to the PARP inhibitor is still high, and the effective duration of the drug is short, so that the use of the PARP inhibitor is limited. Therefore, we hope to further find a method for sensitizing the effect of PARP inhibitors in TNBC. Through previous studies, we believe that modulation of the homologous recombination/non-homologous end joining (HR/NHEJ) pathway is one of the most promising directions for sensitizing PARP inhibitors: antagonizes key molecules in HR/NHEJ pathway to induce HR defect of tumor cells, promote NHEJ repair and generate BRCA 1-like mutation, and the combination of the mechanism and PARP inhibitor can raise the treatment sensitivity and expand the application possibility of PARP inhibitor in non-BRCA 1 mutated TNBC.
CXorf56 (chromosome X open reading frame 56 gene) is a novel protein, located in the 4-band of the long arm 2 region of the X chromosome (Xq 24), consisting of 7 exons, highly conserved in multiple species. Annemieke et al have reported that CXorf56 is involved in X-linked intellectual impairment; it was also found in mouse embryos to be associated with abnormal midbrain-hindbrain boundary morphology, forebrain reduction and developmental delay, suggesting that CXorf56 may play a role in brain development. In addition, little is currently known about the function of the gene, and no report on CXorf56 related to cancer is available. To date, it is unclear whether other key molecules are involved in the regulation of PARP inhibitor treatment sensitivity and the specific mechanism of regulation in breast cancer.
Disclosure of Invention
The purpose of the invention is as follows: in earlier studies, we found a new function of the CXorf56 gene and have preliminarily demonstrated its involvement in regulating the repair efficiency of HR/NHEJ in TNBC. Through further exploration, the specific action mechanism of CXorf56 is determined, the sensitization effect of antagonistic CXorf56 on the PARP inhibitor is evaluated, and the feasibility of people who benefit from the PARP inhibitor is expanded. Unexpectedly, the PARP inhibitor is combined with key genes for inducing the HR defect of tumor cells and promoting the repair of NHEJ, so that the drug resistance of the PARP inhibitor can be overcome, and the clinical application of the PARP inhibitor can be expanded. Therefore, we believe that the HR/NHEJ regulatory pathway is currently one of the most promising research directions.
The invention further discovers that KU70 and CXorf56 can be jointly used for enhancing the sensitivity of olaparib in treating triple negative breast cancer, thereby improving the treatment effect.
The technical scheme is as follows: in order to solve the technical problems, the invention provides application of CXorf56 gene or protein in preparing a medicament for treating triple negative breast cancer or a medicament synergist thereof.
The invention also discloses application of the recombinant vector, the recombinant cell and the recombinant protein for over-expressing the CXorf56 gene or knocking down the CXorf56 gene in preparing a medicament for treating triple negative breast cancer or a medicament synergist thereof.
Wherein, the recombinant vector for over-expressing the CXorf56 gene and the KU70 gene is pLenti-CMV-blast plasmid.
Wherein, the recombinant vector for knocking down the CXorf56 gene and the KU70 gene is pLKO.1-puro plasmid.
Wherein the recombinant cell comprises one or more of MDA-MB-231, BT549, SUM1315, ZR751, MCF-7 or T47D.
The invention also comprises the application of the CXorf56 gene and the KU70 gene in preparing a medicament for treating triple negative breast cancer or a medicament synergist thereof.
The invention also comprises application of the CXorf56 protein and the KU70 protein in preparing a medicament for treating triple negative breast cancer or a medicament synergist thereof.
The invention also comprises the application of the recombinant vector for over-expressing and knocking down the CXorf56 gene and the recombinant vector for over-expressing and knocking down the KU70 gene in preparing a medicament for treating triple negative breast cancer or a medicament synergist thereof.
The invention also discloses an application of the recombinant cell for over-expressing and knocking down the CXorf56 gene and the recombinant cell for over-expressing and knocking down the KU70 gene in preparing a medicament or medicament synergist for treating triple negative breast cancer.
The invention also discloses an application of the recombinant protein for over-expressing and knocking down the CXorf56 gene and the recombinant protein for over-expressing and knocking down the KU70 gene in preparing a medicament or medicament synergist for treating triple negative breast cancer.
Further, the sensitivity of TNBC to olaparib can be enhanced by knocking down CXorf56 gene and overexpressing KU70 gene, thereby improving the therapeutic effect of the drug.
Has the beneficial effects that: the invention discovers that the CXorf56 gene can be knocked down to enhance the effect of the breast cancer medicament in treating the triple-negative breast cancer for the first time. Meanwhile, the fact that the sensitivity of olaparib in treating triple-negative breast cancer can be further enhanced by reducing the expression of CXorf56 gene and increasing the expression of KU70 gene, so that the treatment effect is improved.
Drawings
FIG. 1: screening shows that the CXorf56 deletion obviously influences the HR/NHEJ repair efficiency and can be used as a biomarker with research potential.
FIG. 2 is a schematic diagram: CXorf56 was significantly highly expressed in TNBC and correlated with poor prognosis of breast cancer.
FIG. 3: the level of CXorf56 expression in breast cancer tissue correlates with molecular typing, later disease stage, and poor prognosis.
FIG. 4: the effect of the CXorf56 deletion on TNBC cell DNA damage repair efficiency was verified.
FIG. 5: CXorf56 may affect sensitivity to olaparib by modulating HR and NHEJ.
FIG. 6: CXorf56 in combination with KU70 modulates the HR/NHEJ pathway, thereby affecting sensitivity to the drug;
FIG. 7: mouse experimental results;
FIG. 8: comparing the sizes of the tumors in the mouse experiment;
FIG. 9: tumor weight comparison in mouse experiments.
Detailed Description
Experimental materials and instruments:
| Olaparib (AZD2281) | selectk, USA |
| Magnetic bead co-immunoprecipitation kit | ThermoFisher scientific, USA |
| Flag gel beads | Sigma Co Ltd |
| pLKO.1-puro vector | Addgene, USA |
| pLenti-CMV-blast vector | Addgene, USA |
| pBIND vector | Addgene, USA |
| pGL3-Basic vector | Promega Corp, USA |
| IgG antibody (ab 37415) | Abcam corporation, UK |
| HA antibody (1:2000, 26183) | Invitrogen corporation of USA |
| FLAG antibody (1:2000, ab205606) | Abcam corporation, UK |
| Beta-actin antibody (1:5000, sc-81178) | Santa Cruz, USA |
| Protein A/G PLUS-Agarose | Santa Cruz USA |
| pDRGFP(#26475) | Addgene, USA |
| pimEJ5GFP（#44026） | Addge Inc. of USA |
| pCMV-N-mCherry(# 123215) | Addge Inc. of USA |
| pCBA-SceI（#26477） | Addge Inc. of USA |
| γH2AX（1:1000，#7631） | CST Corp, USA |
| KU70（1:1000，#4588） | CST Corp, USA |
| MDC1（1:1000，ab50003） | Abcam corporation of America |
| 53BP1（1:1000，#4937） | CST Corp, USA |
| BRCA1（1:1000，#9010） | CST Corp, USA |
| RPA（1:1000，sc56770） | Santa Cruz, usa |
| RAD51（1:1000，#8875） | CST Corp, USA |
| CXorf56 knock-down model (shCX) | BEIJING SYNGENTECH Co.,Ltd. |
| KU70 knock-down model (shKU 70) | BEIJING SYNGENTECH Co.,Ltd. |
| CXorf56 overexpression model (Rescued 1 and Rescued 2) | BEIJING SYNGENTECH Co.,Ltd. |
The sequence related by the invention is as follows:
siRNA sequences：
siRNA sequences：
si-NTC (control)
SEQ ID NO:1:Sense: 5’-GCACAAGCUGGAGUACAACUACATT-3’
SEQ ID NO:2:Anti-sense: 5’-UGUAGUUGUACUCCAGCUUGUGCTT-3’
si-CXorf56#1:
SEQ ID NO:3:Sense: 5’-ACAAACAGUAGUAAACAUGGA-3’
SEQ ID NO:4:Anti-sense: 5’-CAUGUUUACUACUGUUUGUGC-3’
si-CXorf56#2:
SEQ ID NO:5:Sense: 5’-UAAAUGGACUCAGUUAAAGAC-3’
SEQ ID NO:6:Anti-sense: 5’-CUUUAACUGAGUCCAUUUAAC-3’
si-DOT1L#1:
SEQ ID NO:7:Sense: 5’-AAUUAAAACGUAAUUCUCCAU-3’
SEQ ID NO:8:Anti-sense: 5’-GGAGAAUUACGUUUUAAUUGA-3’
si-DOT1L#2:
SEQ ID NO:9:Sense: 5’-UUUCUAAGGAGAUUGUUGCAA-3’
SEQ ID NO:10:Anti-sense: 5’-GCAACAAUCUCCUUAGAAAGC-3’
si-NFKBIA#1:
SEQ ID NO:11:Sense: 5’-UUGUACAAAUAUACAAGUCCA-3’
SEQ ID NO:12:Anti-sense: 5’-GACUUGUAUAUUUGUACAAAA-3’
si-NFKBIA#2:
SEQ ID NO:13:Sense: 5’-AUCUGUUUAAUAAAUAUACAU-3’
SEQ ID NO:14:Anti-sense: 5’-GUAUAUUUAUUAAACAGAUUU-3’
si-TAF8#1:
SEQ ID NO:15:Sense: 5’-UAUUCUGUUCAUAUCUUCCUC-3’
SEQ ID NO:16:Anti-sense: 5’-GGAAGAUAUGAACAGAAUAAU-3’
si-TAF8#2:
SEQ ID NO:17:Sense: 5’-AUGUUUCAAGUUGUCUAACCC-3’
SEQ ID NO:18:Anti-sense: 5’-GUUAGACAACUUGAAACAUUG-3’
si-GTF2H5#1:
SEQ ID NO:19:Sense: 5’-UAUAAGCACUCCUUUCAAGAC-3’
SEQ ID NO:20:Anti-sense: 5’-CUUGAAAGGAGUGCUUAUAGA-3’
si-GTF2H5#2:
SEQ ID NO:21:Sense: 5’-AUACAGAUGGGAAAAUGUGAC-3’
SEQ ID NO:22:Anti-sense: 5’-CACAUUUUCCCAUCUGUAUUC-3’
si-BCAP31#1:
SEQ ID NO:23:Sense: 5’-AAUGAGAACCACAAAGAAGGU-3’
SEQ ID NO:24:Anti-sense: 5’-CUUCUUUGUGGUUCUCAUUGU-3’
si-BCAP31#2:
SEQ ID NO:25:Sense: 5’-AUCAUACUUCCGAAUUUCGCG-3’
SEQ ID NO:26:Anti-sense: 5’-CGAAAUUCGGAAGUAUGAUGA-3’
si-SLC31A1#1:
SEQ ID NO:27:Sense: 5’-AUUCUUAAAGCCAAAGUAGAA-3’
SEQ ID NO:28:Anti-sense: 5’-CUACUUUGGCUUUAAGAAUGU-3’
si-SLC31A1#2:
SEQ ID NO:29:Sense: 5’-AAGUCAUUGAACAAAAAGCUA-3’
SEQ ID NO:30:Anti-sense: 5’-GCUUUUUGUUCAAUGACUUGA-3’
si-GDPD2#1:
SEQ ID NO:31:Sense: 5’-AAUAGCAAGGAUGCAUAUGUG-3’
SEQ ID NO:32:Anti-sense: 5’-CAUAUGCAUCCUUGCUAUUGG-3’
si-GDPD2#2:
SEQ ID NO:33:Sense: 5’-UGUUCUAGUUUCAUUUGAGGA-3’
SEQ ID NO:34:Anti-sense: 5’-CUCAAAUGAAACUAGAACAGA-3’
si-ZDHHC9#1:
SEQ ID NO:35:Sense: 5’-AGAACAAAGAUCAAAAUCCGU-3’
SEQ ID NO:36:Anti-sense: 5’-GGAUUUUGAUCUUUGUUCUUC-3’
si-ZDHHC9#2:
SEQ ID NO:37:Sense: 5’-UACUUACAUAUAUCUAAUGGG-3’
SEQ ID NO:38:Anti-sense: 5’-CAUUAGAUAUAUGUAAGUAGU-3’
si-MCTS1#1:
SEQ ID NO:39:Sense: 5’-UAAAAGGAUAUUUGUGAAGUA-3’
SEQ ID NO:40:Anti-sense: 5’-CUUCACAAAUAUCCUUUUAUC-3’
si-MCTS1#2:
SEQ ID NO:41:Sense: 5’-UAUCAUUAGCCUAAGAAAGGA-3’
SEQ ID NO:42:Anti-sense: 5’-CUUUCUUAGGCUAAUGAUAUA-3’
Conventional qPCR primer sequences:
CXorf56:
SEQ ID NO:43:Forward: 5’-ACTGTCAGAGGAGACCCATTA-3’
SEQ ID NO:44:Reverse: 5’-CCTCCCAAACTGCTAGGATTAC-3’
DOT1L:
SEQ ID NO:45:Forward: 5’-ACATTGGAGAGAGGCGATTTC-3’
SEQ ID NO:46:Reverse: 5’-CAAGTATGGTGCGGTCGATAG-3’
NFKBIA:
SEQ ID NO:47:Forward: 5’-CATCCTGAAGGCTACCAACTAC-3’
SEQ ID NO:48:Reverse: 5’-CTCTGTGAACTCCGTGAACTC-3’
TAF8:
SEQ ID NO:49:Forward: 5’-CCTCCCAAAGTGCTGAGATTAC-3’
SEQ ID NO:50:Reverse: 5’-GGCCAGATGAGGAGAGAAATAAG-3’
GTF2H5:
SEQ ID NO:51:Forward: 5’-GTCGAAACCCTGTCTCTACAAA-3’
SEQ ID NO:52:Reverse: 5’-TCCCAAAGTGCTGGGATTAC-3’
BCAP31:
SEQ ID NO:53:Forward: 5’-GCCCAGAGGAATCTCTACATTG-3’
SEQ ID NO:54:Reverse: 5’-CCTGTTCTCTTCCTCCAACTTC-3’
SLC31A1:
SEQ ID NO:55:Forward: 5’-GGGAAATTCTTGCCCAACTAAAC-3’
SEQ ID NO:56:Reverse: 5’-CCAGACACACCATCTACCAATC-3’
GDPD2:
SEQ ID NO:57:Forward: 5’-CCAGATGAAGATCGGGCTAATG-3’
SEQ ID NO:58:Reverse: 5’-CACACCAAAGCAGAGAGAAGAG-3’
ZDHHC9:
SEQ ID NO:59:Forward: 5’-CTCTCTCCCTCCTCACAATCTA-3’
SEQ ID NO:60:Reverse: 5’-CAGCTTACTTGCTCTCTGTCTC-3’
MCTS1:
SEQ ID NO:61:Forward: 5’-AGCAAATCCCAGACATCCTATC-3’
SEQ ID NO:62:Reverse: 5’-CAGGTCCATGTCCCTCATATTC-3’
GAPDH:
SEQ ID NO:63:Forward: 5’-AGAAGGCTGGGGCTCATTTG-3’
SEQ ID NO:64:Reverse: 5’-AGGGGCCATCCACAGTCTTC-3’
shRNA sequence (cloned into pLKO.1-puro vector)
sh-NTC:
SEQ ID NO:65:
Sense: 5’-CCGGTGGTTTACATGTTTTCTGACTCGAGTCAGAAAACATGTAAA
CCATTTTTG-3’
SEQ ID NO:66:
Anti-sense: 5’-AATTCAAAAATGGTTTACATGTTTTCTGACTCGAGTCAGAAA
ACATGTAAACCA-3’
sh-CXorf56#1: SEQ ID NO:67:
Sense: 5’-CCGGGGGAATCATGCCGAAAGTATTCAAGAGATACTTTCGGCATG
ATTCCCTTTTTT-3’
SEQ ID NO:68:
Anti-sense: 5’-AATTAAAAAAGGGAATCATGCCGAAAGTATCTCTTGAATACTT
TCGGCATGATTCCC-3’
sh-CXorf56#2: SEQ ID NO:69:
Sense: 5’-CCGGGCAGAGGAGTGTTGTATATTTCAAGAGAATATACAACACTC
CTCTGCTTTTTT-3’
SEQ ID NO:70:
Anti-sense: 5’-AATTAAAAAAGCAGAGGAGTGTTGTATATTCTCTTGAAATATA
CAACACTCCTCTGC-3’
sh-Ku70: SEQ ID NO:71:
Sense: 5’-CCGGGGTGGGAGTCATATTACAATTCAAGAGATTGTAATATGACTC
CCACCTTTTTT-3’
SEQ ID NO:72:
Anti-sense: 5’-AATTAAAAAAGGTGGGAGTCATATTACAATCTCTTGAATTGTA
ATATGACTCCCACC-3’
Primer sequence adopted by overexpression vector (pLenti-CMV-blast plasmid)
pLenti-CMV-blast-Cxorf56: SEQ ID NO:73:
Forward: 5’-GGGGATCCGCCACC atgaggccccgggaccggtccc-3’
SEQ ID NO:74:
Reverse: 5’-ctcgagCTTGTCGTCATCGTCTTTGTAGTCtttgaactggttgt
caatcaaggt-3a
pLenti-CMV-blast-Ku70-WT: SEQ ID NO:75:
Forward: 5’-GGGGATCCGCCACCatgtcagggtgggagtcatattac-3’
SEQ ID NO:76:
Reverse: 5’-ctcgagCTTGTCGTCATCGTCTTTGTAGTCgtcctggaagtgct
tggtgaggg-3’
pLenti-CMV-blast-Ku70-vWa-domain: SEQ ID NO:77:
Forward: 5’-GGGGATCCGCCACCatgtcagggtgggagtcatattac-3’
SEQ ID NO:78:
Reverse: 5’-ctcgagCTTGTCGTCATCGTCTTTGTAGTCaaagtgaaccctga
ggtcctcatcc-3’
pLenti-CMV-blast-Ku70-DNA-binding-domain: SEQ ID NO:79:
Forward: 5’-GGGGATCCGCCACC GAGGAATCCAGCAAGCTAGAAG-3’
SEQ ID NO:80:
Reverse: 5’-ctcgagCTTGTCGTCATCGTCTTTGTAGTCaaagaccagctgga
agcctggagg-3’
pLenti-CMV-blast-Ku70-C-terminal-domain: SEQ ID NO:81:
Forward: 5’-GGGGATCCGCCACC TTACCCTTTGCTGATGATAAAAGG-3’
SEQ ID NO:82:
Reverse: 5’-ctcgagCTTGTCGTCATCGTCTTTGTAGTCgtcctggaagtgct
tggtgaggg-3’；
The sequences involved in the invention are synthesized by Beijing Synbiotic Gene science and technology Co.
The invention searches a new target point of sensitization of curative effect by exploring a drug resistance mechanism of the PARP inhibitor, and is an important way for improving the effective rate of treatment, expanding benefitting population and improving the survival of patients.
Combining the gene map of DNA damage response and the first 500 genes most related to the total survival (OS) of the primary breast cancer in a TCGA-BRCA database, screening candidate genes which participate in DNA damage repair and influence prognosis, verifying the expression of the candidate genes in the breast cancer and the regulation and control effect on HR/NHEJ, and establishing a target gene (CXorf 56) of the research;
the specific action mechanism of the target gene (CXorf 56) in the PARP inhibitor drug resistance process is discussed.
890 DNA damage related genes are obtained according to a DNA damage response gene map published in Cell; download the first 500 Genes Most related to total Survival (OS) of primary breast cancer (Most Differential Survival Genes) using the GEPIA2 database (http:// GEPIA. cancer-pku. cn/index. html);
the results of the two screening are overlapped to obtain 10 candidate genes, which are shown as a picture B in figure 1 and comprise MCTS1, GDPD2, ZDHHC9, TAF8, SLC31A1, BCAP31, GTF2H5, DOT1L, CXorf56 and NFKBIA.
Example 1 establishment of CXorf56 Gene
Selecting 3 TNBC cell strains (MDA-MB-231, BT549 and SUM 1315) and 3 non-TNBC cell strains (ZR 751, MCF-7 and T47D) (all purchased from ATCC cell bank) as experimental objects; respectively detecting the expression of 10 candidate genes in the cell strain by using RT-PCR, standardizing the result and comparing the expression conditions of the candidate genes; firstly, constructing the 10 candidate gene knockdown cell models by transfecting lipofectamine2000 and corresponding siRNA (the sequence is as described above) in MDA-MB-231, BT549, SUM1315, ZR751, MCF-7 and T47D cells, and secondly, transfecting the last step-knockdown cell models by respectively transfecting a homologous recombination repair pathway specific plasmid pDRGFP or a non-homologous recombination repair pathway specific plasmid pimEJ5 GFP; then transfecting pCMV-N-mCherry plasmid or pCBA-SceI plasmid in the cell line by using lipofectamine2000 reagent, and detecting the percentage of GFP positive cells in the cells by using a flow cytometer, wherein the percentage represents the HR and NHEJ repair efficiency; the results of repair efficiency were normalized and compared to the effect of candidate gene knockdown on HR/NHEJ, with CXorf56 deletion having the greatest effect on HR/NHEJ, cells with CXorf56 deletion showing HR repair defects and increased levels of NHEJ repair, which was more pronounced in TNBC. See figure 1 for experimental procedures and results. CXorf56 was therefore established as the gene of interest.
Example 2 correlation study of CXorf56 with breast cancer
The TCGA database was used to download the RNA sequencing data (https:// portal.gdc.cancer. gov /) for breast cancer tissue samples with the tissue sample screening criteria: the primary breast cancer is confirmed through pathology, complete clinical data and follow-up results are obtained, and 1068 primary breast cancer tissue samples are finally obtained for subsequent analysis; expression levels of CXorf56 in each molecular subtype were analyzed and compared and correlated with prognosis of primary breast cancer.
As can be seen in fig. 2, of these, 831 tissue samples had detailed Immunohistochemical (IHC) data: 122 cases of TNBC, 108 cases of HER2 overexpression type, 601 cases of ER/PR positive type; 512 tissue samples had complete PAM50 typing data: 97 cases of Basal-like model, 58 cases of HER 2-evolved model, 229 cases of Luminal model A, 120 cases of Luminal model B and 8 cases of Normal-like model; (panel a, panel B in figure 2) CXorf56 was significantly more expressed in both TNBC and Basal-like subtypes than in the other subtypes; (panel C in figure 2) with the median CXorf56 expression as the demarcation between high and low expression groups, CXorf56 high expressing patients had poor OS based on Kaplan-Meier survival analysis.
Selecting 180 cases of paraffin specimens of breast cancer patients which are subjected to operation and pathologically confirmed diagnosis in breast surgery of a tumor hospital in Jiangsu province in 2011-2014; all the study objects are fixed by 4% neutral formalin, embedded by normal paraffin, and the thickness of a paraffin section is 4 mu m, and immunohistochemical staining and follow-up are carried out; the expression and clinical significance of CXorf56 in each subtype of breast cancer are analyzed.
Immunohistochemistry results from figure 3 show that: in panel B of fig. 3, CXorf56 is significantly higher in TNBC and lower in non-TNBC; in panel C of fig. 3, CXorf56 is expressed more in stage III breast cancer and less in stage I-II breast cancer according to TNM staging; FIG. 3 is a D-plot showing that CXorf56 is highly expressed in subgroups with tumor maximum diameters of 3cm or more and less expressed in subgroups < 3 cm; FIG. 3, panel E, defines immunohistochemistry scores of 0-3 as low expression of CXorf56 and scores of 4-7 as high expression, suggesting that the high expression subgroup of CXorf56 has poorer OS based on Kaplan-Meier survival analysis; the F plot in fig. 3, multivariate Cox regression analysis, shows whether it is TNBC and CXorf56 expression levels and is an independent prognostic factor for OS.
Example 3 construction of CXorf56 knockdown and overexpression cell models and validation of knockdown efficiency
Synthesis of CXorf56 specific interference sequences (shRNA sequences) and packaging of lentiviruses, lentivirus infection and drug screening, and validation of knockdown efficiency using RT-PCR were performed. Constructing a CXorf 56-knocked-down cell model and an over-expressed cell model (the sequence is as described above, by Synbiotic Gene technology Co., Ltd., Beijing), and constructing the completed cell models for subsequent experiments; cell immunofluorescence assay: gamma H2AX is used as a DNA double-strand break marker, and the change of gamma H2AX foci expression caused by CXorf56 deletion is detected by comparing the number of gamma H2AX foci expression in cells among groups to be more than 10, so that CXorf56 is proved to participate in a DNA damage repair pathway;
panel A, B, C in FIG. 4 were lentivirus infected in MDA-MB-231, BT549 and SUM1315 cells, respectively, demonstrating CXorf56 knockdown efficiency; CXorf56 knockdown was performed on MDA-MB-231, BT549 and SUM1315 cells, and the knocked-down cells were marked as shCXorf56# 1 and shCXorf56# 2 cells, and the control cells infected with unloaded lentivirus were marked as NTC cells; the cells in the IR group are divided into an IR group and a non-treatment group (Ctrl), the cells in the IR group are subjected to 2Gy gamma ray treatment (Beeubao, Germany, irradiation field is 10cm multiplied by 10cm, single dose is 2 Gy), the cells are fixed and subjected to cellular immunofluorescence detection 8 hours after irradiation, the number of cells with gamma H2AX foci expression of more than 10 in the cells is counted, gamma H2AX is used as a DNA double-strand break marker, and the comparison of the number of cells with gamma H2AX foci expression of more than 10 in the cells in each group proves that the deletion of CXorf56 causes the change of gamma H2AX foci expression and participates in a DNA damage repair pathway. The results show that the D, E, F, G, H and I maps in FIG. 4 have no statistical difference in the expression of shCXorf56# 1, shCXorf56# 2 and gamma H2AX foci in NTC cells in MDA-MB-231, BT549 and SUM1315 three TNBC cells respectively; after 2Gy gamma ray irradiation in the IR group, the expression of γ H2AX foci in shCXorf56# 1, shCXorf56# 2 and NTC cells was significantly increased, while the increase of foci in shCXorf56# 1 and shCXorf56# 2 cells was significantly higher than that in NTC cells.
The Western Blot experiment is used for verifying the knocking-down and over-expression efficiency of CXorf56 in a Cxorf56 knocking-down and over-expression cell model; under the treatment conditions of different radiation doses (0, 1, 2, 3, 4 and 5 Gy) and drug concentrations (0, 0.5, 1, 1.5 and 2 mu M), the clone formation rate ratio (clone formation number/inoculated cell number) of the cells after CXorf56 knockdown is detected by using a clone formation experiment so as to reflect the sensitivity change of the cells to gamma rays, platinum and olaparib when CXorf56 is deleted; analyzing the HR and NHEJ repair efficiency of CXorf56 knockdown cells and a control group after gamma ray irradiation by using a DNA damage repair report system, and verifying the regulation and control effect of CXorf56 on HR/NHEJ; performing cell cycle experiments to detect the effect of CXorf56 expression on the cell cycle; detecting whether CXorf56 knockdown cells and a control group influence the expression of key proteins in HR and NHEJ repair pathways after gamma ray irradiation by using an immunofluorescence experiment; the interaction of CXorf56 and a key protein (KU 70) in an HR/NHEJ pathway is verified through a Co-IP experiment, a Co-knockdown cell model is further constructed, and the interaction of CXorf56 and the key protein (KU 70) is confirmed through an immunofluorescence experiment, a clonogenic experiment and a DNA injury repair report system to regulate the treatment sensitivity of TNBC to the PARP inhibitor.
Experimental results referring to fig. 5 and 6, panel a in fig. 5 is a graph demonstrating CXorf56 knockdown and overexpression efficiency using Western blot; panel B in FIG. 5, Panel C in FIG. 5, Panel D in FIG. 5 clonogenic experiments to measure the concentration gradients of CXorf56 knockdown and over-expressed MDA-MB-231 cells on gamma rays, carboplatin and Olaparib, indicating that CXorf56 can affect the sensitivity of MDA-MB-231 cells to radiation and drugs; FIG. 5, panels E and F, analyze the HR and NHEJ repair efficiencies of CXorf56 knockdown and over-expressed MDA-MB-231 cells after receiving 2Gy IR, and cells with different CXorf56 expression levels can generate different HR and NHEJ repair efficiencies after receiving IR, which indicates that CXorf56 participates in regulating HR/NHEJ repair, namely CXorf56 participates in DNA damage repair; the results of the G plot in fig. 5 indicate that inducing competitive inhibition of the NHEJ pathway by the NHEJ pathway at low CXorf56 expression, and that HR enhancement competitively inhibits NHEJ at high CXorf56 expression; FIG. 5 is a H-plot showing that knockdown and overexpression of CXorf56 did not affect the MDA-MB-231 cell cycle; after 2Gy IR treatment of panels I and J in fig. 5, CXorf56 was highly expressed inhibiting KU70 expression while inducing increased expression of BRCA1, RPA and RAD51, demonstrating that CXorf56 high expression might affect sensitivity to treatment by enhancing the HR pathway through inhibition of KU 70. Panel A, B, and C of FIG. 6 demonstrate, by Co-IP assays, that KU70 binds to CXorf56 in MDA-MB-231 cells, and KU70 is a key downstream protein of CXorf 56; the D-map in fig. 6 demonstrates sensitivity to olaparib in the negative control group (NTC), CXorf56 knockdown cell model (shCX), KU70 knockdown cell model (shKU 70), CXorf56 and KU70 co-knockdown cell model (shCX + shKU 70), respectively, using a clonogenic assay, and the results show that CXorf56 is best sensitive to olaparib when expressed low, and that shKU70 and shCX + shKU70 result in drug resistance to olaparib; immunofluorescence experiments in panels E and F in fig. 6 confirmed that γ H2AX foci expression was highest in shCX cells after olaparib treatment (1.5 uM), which shCX + shKU70 reversed; in the G and H plots of fig. 6, NHEJ efficiency was increased and HR efficiency was decreased in shCX cells using the DNA damage repair system, and shCX + shKU70 reversed this phenomenon. The above results all indicate that the interaction of CXorf56 with the key protein KU70 is involved in DNA damage repair and TNBC sensitivity to PARP inhibitor treatment.
Example 4 mouse experiments
The effect of CXorf56 and KU70 on olaparib drugs in the MDA-MB-231 cell nude mouse subcutaneous neoplasia model was compared. As can be seen from fig. 7, 8, and 9, the nude mouse model of shKU70 subcutaneous neoplasia had increased sensitivity to olaparib compared to the negative control group under olaparib concentration of 1.5uM (nude mouse gavage), but was lower than the nude mouse model of shCX subcutaneous neoplasia; the shCX subcutaneous tumorigenic model has the highest sensitivity to Olaparib, the tumor growth is obviously inhibited, the tumor volume and the mass are minimum, the shCX + shKU70 cell subcutaneous tumorigenic model reverses the phenomenon, the sensitivity to Olaparib is obviously reduced compared with the shCX group, and the tumor volume and the mass are larger; this phenomenon also demonstrates that inhibition of CXorf56 expression can enhance the sensitivity of triple negative breast cancer to olaparib, and KU70 may be involved in functioning as a downstream key protein of CXorf 56.
Sequence listing
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Claims (5)
1. The recombinant vector, the recombinant cell and the recombinant protein for knocking down the CXorf56 gene are applied to the preparation of a medicament synergist for treating triple negative breast cancer.
2. The use according to claim 1, wherein the recombinant vector for knocking down the CXorf56 gene is a pLKO.1-puro plasmid.
3. The recombinant vector for knocking down CXorf56 gene and the recombinant vector for over-expressing KU70 gene are applied to the preparation of a drug synergist for treating triple negative breast cancer.
4. Application of recombinant cells for knocking down CXorf56 gene and recombinant cells for over-expressing KU70 gene in preparation of drug synergist for treating triple negative breast cancer.
5. Application of recombinant protein for knocking down CXorf56 gene and recombinant protein for over-expressing KU70 gene in preparing medicine synergist for treating triple negative breast cancer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3260552A1 (en) * | 2016-06-20 | 2017-12-27 | Istituto Europeo di Oncologia (IEO) | Methods and kits comprising gene signatures for stratifying breast cancer patients |
| WO2018204764A1 (en) * | 2017-05-05 | 2018-11-08 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
| CN111328287A (en) * | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | Novel nucleic acid molecules |
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| EP3548635B1 (en) * | 2016-11-30 | 2021-04-28 | Exosome Diagnostics, Inc. | Methods to detect mutations in plasma using exosomal rna and cell free dna from non-small cell lung cancer patients |
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| EP3260552A1 (en) * | 2016-06-20 | 2017-12-27 | Istituto Europeo di Oncologia (IEO) | Methods and kits comprising gene signatures for stratifying breast cancer patients |
| WO2018204764A1 (en) * | 2017-05-05 | 2018-11-08 | Camp4 Therapeutics Corporation | Identification and targeted modulation of gene signaling networks |
| CN111328287A (en) * | 2017-07-04 | 2020-06-23 | 库瑞瓦格股份公司 | Novel nucleic acid molecules |
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| novel clinical and genetic insight into CXorf56-associated intellectual disability;Rocha等;《European journal of human genetics》;20201231;第28卷(第3期);第367-372页 * |
| STEEP mediates STING ER exit and activation of signaling;Zhang BC等;《nature immunology》;20200731;第21卷(第8期);第868页 * |
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