CN112933110A - Marker for sensitivity of lung cancer patient to chemotherapeutic drugs and application thereof - Google Patents
Marker for sensitivity of lung cancer patient to chemotherapeutic drugs and application thereof Download PDFInfo
- Publication number
- CN112933110A CN112933110A CN202110148835.1A CN202110148835A CN112933110A CN 112933110 A CN112933110 A CN 112933110A CN 202110148835 A CN202110148835 A CN 202110148835A CN 112933110 A CN112933110 A CN 112933110A
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- baicalin
- cisplatin
- marker
- xrcc1
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010058467 Lung neoplasm malignant Diseases 0.000 title claims abstract description 64
- 201000005202 lung cancer Diseases 0.000 title claims abstract description 64
- 208000020816 lung neoplasm Diseases 0.000 title claims abstract description 64
- 239000002246 antineoplastic agent Substances 0.000 title claims abstract description 23
- 229940044683 chemotherapy drug Drugs 0.000 title claims abstract description 21
- 239000003550 marker Substances 0.000 title claims abstract description 17
- 230000035945 sensitivity Effects 0.000 title claims abstract description 15
- IPQKDIRUZHOIOM-UHFFFAOYSA-N Oroxin A Natural products OC1C(O)C(O)C(CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IPQKDIRUZHOIOM-UHFFFAOYSA-N 0.000 claims abstract description 51
- AQHDANHUMGXSJZ-UHFFFAOYSA-N baicalin Natural products OC1C(O)C(C(O)CO)OC1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 AQHDANHUMGXSJZ-UHFFFAOYSA-N 0.000 claims abstract description 51
- IKIIZLYTISPENI-ZFORQUDYSA-N baicalin Chemical compound O1[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1OC(C(=C1O)O)=CC2=C1C(=O)C=C(C=1C=CC=CC=1)O2 IKIIZLYTISPENI-ZFORQUDYSA-N 0.000 claims abstract description 51
- 229960003321 baicalin Drugs 0.000 claims abstract description 51
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims abstract description 49
- 229960004316 cisplatin Drugs 0.000 claims abstract description 49
- 101150042435 Xrcc1 gene Proteins 0.000 claims abstract description 34
- 230000014509 gene expression Effects 0.000 claims abstract description 12
- 229930014626 natural product Natural products 0.000 claims abstract description 8
- 229940122498 Gene expression inhibitor Drugs 0.000 claims abstract description 4
- 230000005764 inhibitory process Effects 0.000 claims abstract description 3
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 2
- 108020004459 Small interfering RNA Proteins 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims description 2
- 239000004055 small Interfering RNA Substances 0.000 claims description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims description 2
- 229940127089 cytotoxic agent Drugs 0.000 claims 3
- 239000003814 drug Substances 0.000 abstract description 23
- 229940079593 drug Drugs 0.000 abstract description 20
- 230000000694 effects Effects 0.000 abstract description 13
- 238000001514 detection method Methods 0.000 abstract description 6
- 230000035572 chemosensitivity Effects 0.000 abstract description 2
- 238000003745 diagnosis Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 72
- 239000003560 cancer drug Substances 0.000 description 30
- 230000006907 apoptotic process Effects 0.000 description 18
- 108010000443 X-ray Repair Cross Complementing Protein 1 Proteins 0.000 description 14
- 102000002258 X-ray Repair Cross Complementing Protein 1 Human genes 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 12
- 230000030279 gene silencing Effects 0.000 description 12
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- 238000012226 gene silencing method Methods 0.000 description 9
- 229910052697 platinum Inorganic materials 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 7
- 238000002512 chemotherapy Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 229920000936 Agarose Polymers 0.000 description 6
- 230000009471 action Effects 0.000 description 6
- 239000000853 adhesive Substances 0.000 description 6
- 230000001070 adhesive effect Effects 0.000 description 6
- 238000003927 comet assay Methods 0.000 description 6
- 231100000170 comet assay Toxicity 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 238000002844 melting Methods 0.000 description 6
- 230000002018 overexpression Effects 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000007035 DNA breakage Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000013467 fragmentation Methods 0.000 description 5
- 238000006062 fragmentation reaction Methods 0.000 description 5
- 102000000412 Annexin Human genes 0.000 description 4
- 108050008874 Annexin Proteins 0.000 description 4
- 102000006382 Ribonucleases Human genes 0.000 description 4
- 108010083644 Ribonucleases Proteins 0.000 description 4
- 239000012148 binding buffer Substances 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000003208 gene overexpression Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 108090000672 Annexin A5 Proteins 0.000 description 3
- 102000004121 Annexin A5 Human genes 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 239000002547 new drug Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 238000010839 reverse transcription Methods 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 108050005848 Annexin A10 Proteins 0.000 description 2
- 102100028117 Annexin A10 Human genes 0.000 description 2
- 230000005971 DNA damage repair Effects 0.000 description 2
- 108060004795 Methyltransferase Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- 230000033616 DNA repair Effects 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 208000037323 Rare tumor Diseases 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000003915 air pollution Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000033590 base-excision repair Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- -1 cells Substances 0.000 description 1
- 239000002962 chemical mutagen Substances 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000005337 ground glass Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000033443 single strand break repair Effects 0.000 description 1
- 239000000779 smoke Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/243—Platinum; Compounds thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a marker for sensitivity of a lung cancer patient to chemotherapeutic drugs and application thereof, belonging to the technical field of biomedical detection and diagnosis, wherein the marker is prepared from chemotherapeutic drugs and natural products, the chemotherapeutic drugs are cisplatin, the natural products are baicalin, and the chemosensitivity of lung cancer cells to cisplatin, baicalin and combined drugs thereof can be effectively improved by targeted interference of an XRCC1 gene expression inhibitor and inhibition of XRCC1 gene expression, so that the administration dosage of the cisplatin, baicalin and combined drugs thereof can be reduced, and the side effects of the cisplatin and baicalin to organisms can be reduced.
Description
Technical Field
The invention belongs to the technical field of biomedical detection and diagnosis, and particularly relates to a relation between an XRCC1 gene and the sensitivity of anti-lung cancer chemotherapy drugs.
Background
Epidemiology indicates that lung cancer is one of the most prevalent malignancies in the world today. In China, because China is in the stage of industrialization and urbanization, air pollution, incapability of effectively controlling smoke and other factors, lung cancer is changed from the past rare tumors into the first tumors with the morbidity and the mortality. Therefore, reducing the morbidity and mortality of lung cancer is an important issue for medical workers in China.
Cisplatin is a common chemotherapy medicament for treating lung cancer clinically, can be combined with DNA to form cross connection, thereby destroying the structure and function of the DNA, inhibiting RNA and protein synthesis, being effective on solid tumors such as lung cancer, breast cancer and the like, greatly improving the life quality of patients and prolonging the life cycle; the occurrence of cisplatin drug resistance limits the clinical application of cisplatin, and the development of new chemotherapeutic drugs becomes a necessary trend along with the progress of cancer prevention and treatment technologies, and natural products have important effects in the development of new drugs because of containing various chemical and biological active anticancer groups.
On one hand, chemotherapy sensitive genes are searched, and on the other hand, new drug development is searched; however, the use of new drugs will also become resistant, and therefore chemotherapy-sensitive genes are sought that are associated with both cisplatin and natural product anti-cancer sensitivity.
Research shows that the down-regulation of the expression of the DNA damage repair gene in the tumor tissue can reduce the contact between the platinum drug and the DNA compound and increase the chemotherapy sensitivity, which is probably one of the factors for improving the survival and prognosis after the chemotherapy of the platinum drug. Therefore, the research of the biomarker prediction and prognosis factor related to the tumor cell is a research hotspot in the field of tumor treatment at present.
XRCC1 is a DNA damage repair gene whose primary function is to participate in base excision repair and DNA single strand break repair of DNA damage caused by ionizing radiation and chemical mutagens. The current research on the XRCC1 gene mainly focuses on the structural polymorphism of the gene. The XRCC1 gene polymorphism is related to tumor susceptibility of almost all tissues and organs in the whole body. In addition, multiple studies have shown that XRCC1 has an increased sensitivity to DNA repair function with reduced sensitivity to chemotherapeutic drugs, and thus the XRCC1 gene may be implicated in the sensitivity of patients to platinum-based drugs for chemotherapy.
Disclosure of Invention
The invention aims to provide a marker for sensitivity of a lung cancer patient to chemotherapeutic drugs and application thereof, and solves the problems that the chemotherapy sensitivity of cisplatin and baicalin to lung cancer is improved in the background art, so that the administration concentration of cisplatin and baicalin is reduced, and the side effect of cisplatin and baicalin to an organism is finally reduced.
In order to achieve the purpose, the invention provides the following technical scheme: a marker for sensitivity of lung cancer patients to chemotherapeutic drugs is prepared from chemotherapeutic drugs and natural products, wherein the chemotherapeutic drugs are cisplatin, and the natural products are baicalin.
Preferably, the lung cancer is non-small cell lung cancer.
Preferably, the target interferes with XRCC1 gene expression.
Preferably, the target interferes with and inhibits XRCC1 gene expression.
Preferably, the XRCC1 gene expression inhibitor targets interference and inhibits XRCC1 gene expression.
Preferably, the XRCC1 gene expression inhibitor comprises siRNA and shRNA.
The application of a marker for susceptibility of a lung cancer patient to a chemotherapeutic drug comprises a marker for susceptibility of a lung cancer patient to a chemotherapeutic drug.
Compared with the prior art, the invention has the beneficial effects that: by reducing the expression of the gene XRCC1 in the lung cancer drug-resistant cell strain, the chemosensitivity of the lung cancer cell to cisplatin, baicalin and combined drugs thereof can be obviously improved;
the expression inhibitor of the gene provided by the invention can effectively improve the chemical sensitivity of lung cancer cells to cisplatin, baicalin and combined medicaments thereof, thereby reducing the administration dosage of the cisplatin, the baicalin and the combined medicaments thereof and simultaneously reducing the side effect of the cisplatin and the baicalin on organisms.
Drawings
FIG. 1 is a graph showing the effect of baicalin on cisplatin apoptosis in lung cancer drug-resistant cell lines detected by flow cytometry;
FIG. 2 is a graph showing the effect of PCR detection of baicalin and cisplatin on drug-resistant cell line XRCC1 of the lung cancer drug-resistant cell line;
FIG. 3 is a graph showing the effect of over-expression of XRCC1 on apoptosis of drug-resistant cell line of lung cancer treated by combination of baicalin and cisplatin by flow cytometry;
FIG. 4 is a graph showing the effect of XRCC1 silencing on apoptosis of drug resistant cell lines of lung cancer treated with a combination of baicalin and cisplatin by flow cytometry;
FIG. 5 is a diagram of the comet assay of the present invention for detecting the effect of XRCC1 overexpression on DNA fragmentation of drug-resistant cell line of lung cancer with combined effect of baicalin and cisplatin;
FIG. 6 is a diagram of the comet assay of the present invention for detecting the effect of XRCC1 silencing on DNA fragmentation of drug resistant cell lines of lung cancer combined with baicalin and cisplatin.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to the attached drawings 1-6, the invention provides a marker for susceptibility of lung cancer patients to chemotherapeutic drugs and application thereof.
The experiment adopts a flow cytometer to detect the apoptosis rate of the baicalin on cisplatin on the lung cancer drug-resistant cell strain. Baicalin or cisplatin is used alone to act on the lung cancer drug-resistant cell strain, or baicalin and cisplatin are combined to act on the lung cancer drug-resistant cell strain, and data detection is carried out by a flow cytometer.
Experiment 1:
inoculating cells into a 100mm culture dish, allowing the cells to adhere to the wall for 24h, and adding baicalin and cis-platinum for treatment for 48 h.
After treatment, trypsinize the cells → centrifugation → collect the cells in flow tube → wash the cells 1 times with PBS (phosphate buffered saline, pH 7.4, isotonic with human blood, major components are sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and potassium chloride);
on ice, 1 × Annexin-binding buffer (Annexin binding buffer) is prepared and resuspended ("resuspension", specifically, solid (precipitate, cells, active substance, etc.) obtained by centrifugation or sedimentation or the like is resuspended in an appropriate buffer or culture solution) cells. Add 5. mu.l Annexin V (a reagent for detecting cell apoptosis) and 10. mu.l PI into the resuspended cells, incubate for 5 minutes at room temperature in the dark, and detect cell apoptosis on a flow cytometer.
The results are shown in figure 1, baicalin and cisplatin can both inhibit and apoptosis lung cancer drug-resistant cell lines, and baicalin can promote apoptosis of cisplatin on lung cancer drug-resistant cell lines.
Experiment 2:
in the experiment, PCR (polymerase chain reaction) is adopted to detect the expression of the combination of single drug of baicalin and cisplatin and the drug-resistant cell strain XRCC1 of the lung cancer. Baicalin and cisplatin jointly act on the lung cancer drug-resistant cell strain, and the detection is carried out by PCR.
The method comprises the following steps:
inoculating cells into a culture dish with the thickness of 100mm, and adding baicalin and cis-platinum for treatment for 48 hours after the cells adhere to the wall for 24 hours; the process after treatment comprises the following steps: trypsinize the cells → centrifuge → collect the cells → extract the RNA.
1. The reverse transcription reagent comprises nuclease-free pure water (RNase free ddH2O), reverse transcription Reaction Buffer (Reaction Buffer), Random Primer, dNTP Mix, RevertAid RT and RiboLock RI.
2. Reverse transcription reagent each component use table
| Composition of matter | Use amount (volume) |
| Reaction Buffer | 4μl |
| Random Primer | 1μl |
| dNTP Mix | 2μl |
| RevertAid RT | 1μl |
| RiboLock RI | 1μl |
3. Mu.g of RNA was added and RNase free ddH2O was added to 20. mu.l.
4. Reaction conditions are as follows: heating at 25 deg.C for 5 min, at 42 deg.C for 60 min, at 70 deg.C for 5 min, and at 4 deg.C to finish.
Amplification:
1. the amplification reagents comprise 2XSYBRGreenSupermix, a forward primer, a reverse primer, cDNA and nuclease-free pure water (RNase free ddH 2O).
2. Usage table of components of amplification reagent
| Composition of matter | Use amount (volume) |
| 2X SYBR Green Supermix | 5μl |
| Forward primer | 1μl |
| Reverse primer | 1μl |
| cDNA | 1μl |
RNase free ddH2O was added to 10. mu.l.
4. Reaction conditions are as follows: first, pre-denaturation at 95 ℃ for 3 minutes → heating at 95 ℃ for 10 seconds → heating at 51 ℃ for 30 seconds → heating at 72 ℃ for 30 seconds for a total of 40 cycles. Set the equipment parameters such that the curve 65 ℃ then increases to 95 ℃ at 0.5C for a5 second process; then the temperature is reduced to 4 ℃, the state is maintained for 10 seconds, and finally the plate is read.
The primer sequence is as follows:
the result is shown in figure 2, baicalin and cisplatin can inhibit lung cancer drug-resistant cell line XRCC1, and the inhibition effect of XRCC1 gene is more obvious when the two drugs act together.
Experiment 3:
the experiment adopts a flow cytometer to detect the apoptosis rate of the over-expression of the XRCC1 gene on the drug-resistant cell strains of baicalin and cisplatin for lung cancer. The lung cancer drug-resistant cell strain is subjected to over-expression of the XRCC1 gene and combined action of baicalin and cisplatin alone, and the lung cancer drug-resistant cell strain is subjected to combined action of the XRCC1 gene, the baicalin and the cisplatin alone, and is detected by a flow cytometer.
The method comprises the following steps:
inoculating cells into a 100mm culture dish, after the cells adhere to the wall for 24 hours, after plasmid transfection for 4 hours, replacing a culture medium for culturing for 48 hours, and treating the cells with baicalin and cis-platinum for 48 hours.
The process after the treatment comprises the following steps: trypsinize cells → centrifuge → collect cells in flow tube → wash cells 1 time with PBS;
on ice, 1 × Annexin-binding buffer was prepared and used to resuspend cells; after resuspension, 5. mu.l Annexin V and 10. mu.l PI were added to the cells, incubated at room temperature in the dark for 5 minutes, and apoptosis was detected on a flow cytometer.
The result is shown in figure 3, the apoptosis condition of the lung cancer drug-resistant cell A549/DDP can be remarkably increased by combining the treatment of the cisplatin and the baicalin, and the influence of the combination of the cisplatin and the baicalin on the apoptosis of the lung cancer drug-resistant cell is remarkably reversed after the XRCC1 is over-expressed, so that the apoptosis rate of the lung cancer drug-resistant cell is reduced after the two drugs act.
Experiment 4:
the experiment adopts a flow cytometer to detect the apoptosis rate of XRCC1 gene silencing on baicalin and cisplatin on lung cancer drug-resistant cell strains. Combining an XRCC1 gene silencing vector with a lung cancer drug-resistant cell strain with combined action of baicalin and cisplatin, combining an XRCC1 gene silencing vector with a lung cancer drug-resistant cell strain with combined action of baicalin and cisplatin, and detecting by a flow cytometer.
The method comprises the following steps:
inoculating cells into a 100mm culture dish, after the cells adhere to the wall for 24 hours, after the XRCC1 gene silencing vector is transfected for 4 hours, changing a culture medium to culture for 48 hours, and treating the cells with baicalin and cis-platinum for 48 hours;
after treatment, trypsinize the cells → centrifuge → collect the cells in flow tube → wash the cells 1 time with PBS;
on ice, 1 × Annexin-binding buffer was prepared and used to resuspend cells; adding 5 ul Annexin V and 10 ul PI into the resuspended cells, and incubating for 5 minutes at room temperature in a dark place; detecting apoptosis on a flow cytometer.
The result is shown in figure 4, the apoptosis of the lung cancer drug-resistant cell A549/DDP can be obviously improved after the treatment of the cisplatin and the baicalin, and the influence of the combination of the cisplatin and the baicalin on the apoptosis of the lung cancer drug-resistant cell is obviously promoted after the XRCC1 is silenced, so that the apoptosis rate of the lung cancer drug-resistant cell is higher after the two drugs act.
Experiment 5:
the experiment adopts comet assay to detect the DNA breakage condition of the over-expression of the XRCC1 gene to the drug-resistant cell strain of baicalin and cisplatin to lung cancer. The lung cancer drug-resistant cell strain is detected by combining an XRCC1 gene overexpression vector with baicalin and cisplatin combined action, and the lung cancer drug-resistant cell strain is combined with the XRCC1 gene overexpression vector with baicalin and cisplatin combined action through a comet assay.
The method comprises the following steps:
inoculating cells in a good logarithmic growth phase state in a 35mm culture dish, after 24 hours of adherence, after transfection of an XRCC1 gene overexpression vector for 4 hours, replacing a culture medium for culture for 48 hours, and adding baicalin and cis-platinum to treat the cells for 48 hours; putting the fully-ground glass slide into a 1% normal melting point agarose solution, taking out, airing and placing for 24 hours at 37 ℃; adding 80 μ l of 0.5% low-melting point agarose solution to the first layer of the adhesive, quickly covering the first layer of the adhesive with a glass slide, and placing the first layer of the adhesive at 4 ℃; adding 20 mul of cell suspension into 160 mul of 0.7% low-melting point agarose solution, uniformly mixing, adding 80 mul of cell suspension on a glass slide paved with a second layer of gel, and standing for 15 minutes at 4 ℃; then adding cell lysis solution and helicase in turn, and carrying out electrophoresis for 30 minutes under the conditions of 25V and 300 mA; after electrophoresis, placing the slide in 100ml of neutralization buffer solution in a dark place and standing for 10 minutes; the slide was washed with distilled water, stained with 40. mu.l of ethidium bromide, and the result was observed under a fluorescence microscope.
The result is shown in figure 5 (the picture needs to be properly magnified and observed), the DNA breakage of the lung cancer drug-resistant cell is reduced by the XRCC1 gene overexpression, the DNA breakage of the lung cancer drug-resistant cell can be obviously increased by combining the cisplatin and baicalin treatment, and the DNA breakage effect of the two drugs is obviously reduced by the XRCC1 overexpression.
Experiment 6:
the experiment adopts comet assay to detect the DNA breakage of the XRCC1 gene silencing on the baicalin and cisplatin on the lung cancer drug-resistant cell strain. The XRCC1 gene silencing vector or the combination of baicalin and cisplatin is used for acting on the lung cancer drug-resistant cell strain, and the XRCC1 gene silencing vector, baicalin and cisplatin are used for acting on the lung cancer drug-resistant cell strain, and detection is carried out through a comet assay.
The method comprises the following steps:
inoculating cells in a good logarithmic growth phase state in a 35mm culture dish, after 24 hours of adherence, after transfection of an XRCC1 gene silencing vector for 4 hours, replacing a culture medium for culture for 48 hours, and adding baicalin and cis-platinum to treat the cells for 48 hours; putting the fully-ground sand glass slide into a 1% normal melting point agarose solution, taking out, airing and placing for 24 hours at the temperature of 37 ℃; adding 80 μ l of 0.5% low-melting point agarose solution to the first layer of the adhesive, quickly covering the first layer of the adhesive with a glass slide, and placing the first layer of the adhesive at 4 ℃; adding 20 mul of cell suspension into 160 mul of l 0.7% low-melting point agarose solution, uniformly mixing, adding 80 mul of cell suspension on a glass slide paved with a second layer of gel, and standing for 15 minutes at 4 ℃; then adding cell lysis solution and helicase in turn, and carrying out electrophoresis for 30 minutes under the conditions of 25V and 300 mA; after electrophoresis, placing the slide in 100ml of neutralization buffer solution in a dark place and standing for 10 minutes; subsequently, the slide glass was washed with triple distilled water, stained with 40. mu.l of ethidium bromide, and the result was observed under a fluorescence microscope.
The result is shown in figure 6 (the picture needs to be properly magnified and observed), the DNA fragmentation of the lung cancer drug-resistant cell is increased by XRCC1 gene silencing, the DNA fragmentation of the lung cancer drug-resistant cell can be obviously increased by combining cisplatin and baicalin treatment, and the effect of combining the two drugs on the DNA fragmentation is obviously increased by XRCC1 silencing. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A marker for sensitivity of lung cancer patients to chemotherapeutic drugs is prepared from chemotherapeutic drugs and natural products, wherein the chemotherapeutic drugs are cisplatin, and the natural products are baicalin.
2. The marker for susceptibility to chemotherapeutic agents for use in a patient with lung cancer according to claim 1, wherein said lung cancer is non-small cell lung cancer.
3. The marker of claim 1, wherein the targeted interference with XRCC1 gene expression is for use in a patient with lung cancer who is sensitive to a chemotherapeutic drug.
4. A marker of sensitivity to chemotherapeutic agents for use in lung cancer patients according to claim 1 or 3, wherein the targeted interference inhibits XRCC1 gene expression.
5. A marker for susceptibility of lung cancer patients to chemotherapeutic agents according to claim 1 or 3 wherein the interference and inhibition of XRCC1 gene expression is targeted by an inhibitor of XRCC1 gene expression.
6. The marker of claim 5, wherein the XRCC1 gene expression inhibitor comprises siRNA, shRNA.
7. Use of a marker for susceptibility to chemotherapeutic drugs for lung cancer patients, comprising a marker for susceptibility to chemotherapeutic drugs for lung cancer patients according to any one of claims 1-6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110148835.1A CN112933110A (en) | 2021-02-03 | 2021-02-03 | Marker for sensitivity of lung cancer patient to chemotherapeutic drugs and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110148835.1A CN112933110A (en) | 2021-02-03 | 2021-02-03 | Marker for sensitivity of lung cancer patient to chemotherapeutic drugs and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112933110A true CN112933110A (en) | 2021-06-11 |
Family
ID=76242189
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110148835.1A Pending CN112933110A (en) | 2021-02-03 | 2021-02-03 | Marker for sensitivity of lung cancer patient to chemotherapeutic drugs and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112933110A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050260586A1 (en) * | 2002-03-28 | 2005-11-24 | Demuth Jeffrey P | Method and compositions for the diagnosis and treatment of non-small cell lung cancer using gene expression profiles |
| CN101744832A (en) * | 2010-01-14 | 2010-06-23 | 山东省中医药研究院 | Chinese traditional medicine preparation for reversing multiple medicine resistance of tumor cells and preparation method thereof |
| CN103494838A (en) * | 2013-09-30 | 2014-01-08 | 武汉大学 | Chemotherapy medicine composition |
| CN109200050A (en) * | 2018-09-13 | 2019-01-15 | 淮安亿泰生物科技有限公司 | Application of the alkaloid in terms of reversing lung cancer cisplatin-resistant |
-
2021
- 2021-02-03 CN CN202110148835.1A patent/CN112933110A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050260586A1 (en) * | 2002-03-28 | 2005-11-24 | Demuth Jeffrey P | Method and compositions for the diagnosis and treatment of non-small cell lung cancer using gene expression profiles |
| CN101744832A (en) * | 2010-01-14 | 2010-06-23 | 山东省中医药研究院 | Chinese traditional medicine preparation for reversing multiple medicine resistance of tumor cells and preparation method thereof |
| CN103494838A (en) * | 2013-09-30 | 2014-01-08 | 武汉大学 | Chemotherapy medicine composition |
| CN109200050A (en) * | 2018-09-13 | 2019-01-15 | 淮安亿泰生物科技有限公司 | Application of the alkaloid in terms of reversing lung cancer cisplatin-resistant |
Non-Patent Citations (1)
| Title |
|---|
| ZHIWEI XU，: ""Baicalin attenuates DDP (cisplatin) resistance in lung cancer by downregulating MARK2 and p-Akt"", 《INTERNATIONAL JOURNAL OF ONCOLOGY》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8507195B2 (en) | MiRNAs dysregulated in triple-negative breast cancer | |
| JP5624470B2 (en) | Expression of miRNA and its use in human peripheral blood microvesicles | |
| Ma et al. | LncRNAs as an intermediate in HPV16 promoting myeloid-derived suppressor cell recruitment of head and neck squamous cell carcinoma | |
| Huang et al. | Clinical significance of microRNA 138 and cyclin D3 in hepatocellular carcinoma | |
| CN113201591B (en) | Application of long-chain non-coding RNA and inhibitor thereof in preventing and treating breast cancer | |
| Su et al. | CircRNAs in lung adenocarcinoma: diagnosis and therapy | |
| Jia et al. | MiR-182-5p inhibited proliferation and migration of ovarian cancer cells by targeting BNIP3. | |
| CN110408703A (en) | Colorectal cancer miRNA marker and its application | |
| WO2012082821A2 (en) | Melanoma treatments | |
| US9157080B2 (en) | Compositions and methods for treatment, diagnosis and prognosis of mesothelioma | |
| CN104208723A (en) | Application of miR-638 in resisting acute myelogenous leukemia | |
| Chen et al. | The abnormal expression of MicroRNA-542-3p in hepatocellular carcinoma and its clinical significance | |
| Keramati et al. | Circulating miRNAs can serve as potential diagnostic biomarkers in chronic myelogenous leukemia patients | |
| US20110250192A1 (en) | Use of microrna signatures for assessing risk levels of neuroblastoma patients | |
| CN107893115B (en) | ALKBH1 gene and application of expression product thereof in preparation of kit for diagnosing tumors and drugs for treating tumors | |
| CN112933110A (en) | Marker for sensitivity of lung cancer patient to chemotherapeutic drugs and application thereof | |
| US20130123138A1 (en) | Compositions and methods for prognosis of mesothelioma | |
| CN111733248A (en) | Application of LOC158435 as biomarker for diagnosing and treating laryngeal squamous cell carcinoma | |
| CN111733247A (en) | Application of long-chain non-coding RNA in cancer diagnosis and treatment | |
| Takizawa et al. | In vitro sensitivity to platinum-derived drugs is associated with expression of thymidylate synthase and dihydropyrimidine dehydrogenase in human lung cancer | |
| CN111088357A (en) | Tumor marker for ESCC and application thereof | |
| US20120190729A1 (en) | Mirna inhibition of six1 expression | |
| Nateghi et al. | Expression profiles of miR-93 and miR-330 in iranian patients with chronic lymphocytic leukemia | |
| CN110946872B (en) | Application of miR-4491 in preparation of medicine for treating breast cancer | |
| CN112375823B (en) | Application of miRNA inhibitor in preparation of medicine for treating and/or preventing lymphoma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210611 |