CN102220336A

CN102220336A - Tumour chemotherapy drug sensitive gene and application of same

Info

Publication number: CN102220336A
Application number: CN 201010148616
Authority: CN
Inventors: 杨林; 杨楠; 黄行许; 吴小军; 钱建飞; 杨文荣
Original assignee: 杨林; 黄行许; 吴小军
Current assignee: PersonGen Biomedicine (Suzhou) Co.,Ltd.
Priority date: 2010-04-14
Filing date: 2010-04-14
Publication date: 2011-10-19

Abstract

The invention discloses a gene capable of determining the sensitivity of tumour cells to chemotherapy drugs, which is named TJP1 or ZO-1. The invention also discloses a method for predicting the chemotherapeutic effect on tumour by using the gene, and patients in which TJP1exrpression exists are sensitive to the chemotherapy drugs; and the patients suffering the loss of expression of TJP1 are endowed with tolerance to the chemotherapy drugs. The invention also discloses a method for improving the chemotherapeutic effect by improving or inducing TJP1 expression as well as the application of the gene to the prediction of chemotherapeutic effect on tumour, the increasing of tumour chemotherapy sensitivity and the preparation of oncotherapy drugs. The invention can be beneficial to the development of a TJP1/ZO-1 expression related diagnostic reagent kit so as to predict the chemotherapeutic effect on patients, and also beneficial to the screening of anticarcinogen so as to find out chemical and biological molecules inducing the TJP1/ZO-1 expression, thereby finally achieving the purposes of reducing the times of chemotherapy, increasing the recovery rate, alleviating the toxic side effects of the chemotherapy drugs, saving the hospitalization costs and generating outstanding economic and social benefits.

Description

Tumour chemotherapy medicament sensitive gene and application thereof

Technical field

The present invention relates to a kind of gene and application that the tumour cell chemotherapy drug susceptibility is had decisive action, particularly relate to this gene as target detect with the prediction patient to chemotherapeutics whether responsive and induce or improve this gene in the intravital expression of patient to reach the chemotherapy effect that strengthens patient.

Background technology

The main treatment means of cancer is excision, radiotherapy, chemotherapy and immunological therapy etc.Wherein chemotherapy-both chemotherapy was the whole body therapeutic method of tumour, used extensivelyr, had become the main means for the treatment of malignant tumor, especially for neoplastic hematologic disorder, as leukemia, lymphoma and myelomatosis, and in, the treatment of terminal cancer.But there is very big toxic side effect in tumor chemotherapeutic drug and very easily produces resistance, has greatly limited the use and the result of treatment of chemotherapy.Though there is at present multiple chemotherapeutics to select for the doctor, owing to can't judge susceptibility and the resistance of different tumour patients to medicine before treatment, so the clinician mainly rule of thumb treats the patient, so have suitable blindness.Inappropriate pharmacological agent not only makes patient's normal body cell and immunity system be destroyed; the misery that the patient is treated; main is but that also inducing tumor cell generation is to the associating resistance of multiple medicine; be referred to as multidrug resistance; thereby cause the failure of chemotherapy; make the patient lose the chance of treatment, also make the patient cause heavy financial loss.

As previously mentioned, the generation of tumor cell drug resistance is the major cause of chemotherapy of tumors failure.The neoplastic hematologic disorder (comprising myelomatosis) that is main treatment means for serious dependence chemotherapy particularly solves the reason that resistance produces, the gene that finds the decision chemosensitivity is a urgent and far reaching problem.With the multiple myeloma is example, and in the past decade, multiple myeloma has become application experiment achievement in research in the neoplastic hematologic disorder and successfully obtained the model of result of treatment.In the world, the new drug of existing 4 kinds of treatment myelomatosis obtains listing in succession, comprise ten thousand jade-like stones (Velcade), Thalidomide (Thalidomide), Macrogol Ester plastid Zorubicin (pegylated liposomaldoxorubicin), and Lei Nali polyamines (lenalidomide).Originally those new drugs are used on one's body recurrent or the difficult multiple myeloma patients that takes effect, and also are widely used in now on the first ill person, and demonstrate the obvious treatment effect.Yet although obtained the progress of the above-mentioned encouraging popular feeling, myelomatosis still can't be cured, and in fact all patients will be recurred this malignant disease, and shows the bigger resistance to chemotherapy.The more important thing is, when even medication shows result of treatment in the early stage, in fact the patient's ratio to specific responding property of chemotherapeutics also is limited, such as: clinical observation find ten thousand best jade-like stones of result of treatment also the multiple myeloma patients of only right～41% complete reaction (CR) and partial reaction (PR) Wan Keyu are arranged and the combination therapy of Macrogol Ester plastid Zorubicin also only shows～44% CR and PR.Traditional chemotherapeutics then effect is poorer, only is～4% CR.Here just bring a very big problem, if that when clinically patient being carried out chemotherapy exactly medicament selection mistake appears, will miss patient's best occasion for the treatment, also can add heavy patient's economical load.

Therefore, the Mechanism Study that drug resistance of tumor is taken place just becomes the research focus in carcinobiology field.Still be example with the myelomatosis, multiple myeloma is a kind of malignant plasma cell knurl, mainly occurs in the marrow, accounts for the about 10% of neoplastic hematologic disorder, is the second pernicious neoplastic hematologic disorder that is only second to non-Hodgkin lymphoma.Over the past decade, a large amount of effort has been used to explore the molecule mechanism of myelomatosis chemotherapy resistance, one of them main mechanism is considered to give the credit to the characteristic of sticking of myeloma cell and extracellular matrix and stroma cell, for example: the apoptosis that is suppressed medicine inductive myeloma cell by the cell adhesion of integrin β 1 mediation.The nearest Wnt3 that studies show that plays an important role to the drug resistance (CAM-DR) that is caused by cell adhesion of VLA-6 (integrin α 6/ β 1) mediation, and this inhibition is to realize by myeloma cell's autocrine mode process Wnt/RhoA/ROCK signal path.In addition, by the interaction of integrins and its part, the member of anti-apoptotic bcl-2 family and the expression of multidrug resistance gene-1 are induced, and reply thereby make tumour cell lose chemotherapeutics.Although above-mentioned progress is encouraging, still unclear now is what to the key factor in the drug resistance of tumor generation, particularly those can be used as the gene of new treatment target in the future, and their expression can significantly improve patient's treatment, and can be used for predicting the chemotherapy effect of tumour patient.

Research and product development to drug sensitive gene both at home and abroad also is in the exploratory stage.In recent years, because the appearance in RNA perturbation technique and library thereof makes the searching chemotherapy medicament sensitive gene consist of possibility.But the relevant RNA of utilization perturbation technique system, screen the tumour chemotherapy medicament sensitive gene group on a large scale, at home and abroad also do not appear in the newspapers as yet.We utilize the RNA interference library, 34 genes have been filtered out first in the world to a series of chemotherapy medicament sensitives such as Zorubicin, cis-platinum, Wan Ke, through further checking, determined the key factor whether responsive that be patient of TJP1 expression of gene wherein to multiple chemotherapeutics.TJP1 is a kind of tight junction protein, mainly is expressed in the junction of cell surface and other cell.In our previous work, we find that the expression of target inhibition TJP1 causes the resistance of Hela cell to Zorubicin, cis-platinum, methotrexate, means that the expression of TJP1 can strengthen the susceptibility of tumour cell to chemotherapeutics.Because the main treatment means of hematologic malignancies seriously relies on chemotherapy, and patient is the major cause that causes treating failure to the resistance of medicine, especially myelomatosis patient, can be in the early stage show after the treatment more serious repeatedly, stronger resistance and virulent occurring degree more, make that myelomatosis is the incurable disease that still can't cure so far.Therefore we have carried out gene expression spectrum analysis to the sample of 264 routine myelomatosis clinical patients, find that expression and the patient of TJP1 replys closely related to the treatment of ten thousand jade-like stones.In the ten thousand jade-like stone drug-resistant cell strain RPMI 8226/BR that we set up, the expression of quantitative PCR detection discovery TJP1 is lost fully.And we carry out TJP1 target silence to the myeloma cell line of ten thousand jade-like stone sensitivities, can induce its chemical sproof generation, prove that fully TJP1 is relevant with myeloma cell's chemosensitivity, the disappearance that TJP1 expresses in the patient body can cause patient that the chemotherapeutics that comprises ten thousand jade-like stones, Zorubicin and cis-platinum is produced resistance.

Summary of the invention

The objective of the invention is: a kind of tumour chemotherapy medicament sensitive gene is provided.

Another object of the present invention is: a kind of method of predicting the chemotherapy of tumors effect is provided.

A further object of the present invention is: a kind of method that improves the chemotherapy of tumors susceptibility is provided.

A further object of the present invention is: the application of said gene in prediction chemotherapy of tumors effect, raising chemotherapy of tumors susceptibility, preparation anti-tumor medicine is provided.

Unnamed gene provided by the present invention is TJP1 (tightjunction protein 1), and perhaps ZO-1 (zona occludens-1) is to have the gene of following mRNA or aminoacid sequence and coded protein thereof:

SEQ ID#1 in the sequence table

SEQ ID#2 in the sequence table

SEQ ID#3 in the sequence table

SEQ ID#4 in the sequence table

The sequence of SEQ ID#1 is by 7165 based compositions in the sequence table; The sequence of SEQ ID#2 is made up of 1748 amino acid in the sequence table; The sequence of SEQ ID#3 is by 6925 based compositions; The sequence of SEQ ID#4 is made up of 1668 amino acid in the sequence table.

The invention provides a kind of gene that determines tumour chemotherapy drug susceptibility, tumor chemotherapeutic drug comprises that all have the chemicals of lethal effect to tumour cell.

The invention provides the method that the TJP1 gene is predicted the chemotherapy of tumors effect of using.When having TJP1 to express in the patient body, patient has susceptibility to chemotherapeutics; When in the patient body during TJP1 expression deletion, patient table reveals the resistance to chemotherapeutics.

It is a kind of by improving or inducing the expression of TJP1 to improve the method for chemotherapy effect that the present invention also provides.This method comprises the intravital expression of external source TJP1 gene introducing patient, also comprises all kinds of chemistry or biomolecules are introduced the expression of inducing the natural TJP1 of patient in the body.

The present invention will help to develop the diagnostic kit of expressing at TJP1/ZO-1, with the result of treatment of prediction patient to chemotherapy; Also help the screening of cancer therapy drug to seek the chemistry and the biomolecules of inducing TJP1/ZO-1 to express, finally reach and make the patient reduce the chemotherapy number of times, increase curative ratio, alleviate the toxic side effect of chemotherapeutics, make the patient save medical expense, produce remarkable economical and social benefit.

Description of drawings

The invention will be further described below in conjunction with drawings and Examples:

Fig. 1, Fig. 2 concern description of drawings for the expression of TJP1 is proportionate with the effect of myelomatosis patient after the treatment of ten thousand jade-like stones, wherein NR: invalid group, and R: effectively organize;

Fig. 3 is the expression map analysis of TJP1 in myeloma cell line;

Fig. 4, Fig. 5 are that the myeloma cell is to the Lethal Dose 50 of adriamycin and Platinol cisplatin and the expression negative correlation of TJP1;

Fig. 6 is the expression of TJP1 in ten thousand jade-like stone resistance RPMI, 8226 cells, RPMI 8226 wild-types and the growth curve of drug-resistant type cell under different ten thousand jade-like stone mass actions;

Fig. 7 is the expression of TJP1 in ten thousand jade-like stone resistance RPMI, 8226 cells, and 10nM ten thousand jade-like stones are handled the apoptosis of cell after 24 hours and measured

Fig. 8 is the expression of TJP1 in ten thousand jade-like stone resistance RPMI, 8226 cells, and quantitative PCR is measured the expression of TJP1mRNA.

Fig. 9, Figure 10, Figure 11 suppress to cause the resistance of Hela cell to Zorubicin, cis-platinum and methotrexate after TJP1 expresses for target;

Figure 12 is the expression that TJP1shRNA (shT2) has effectively suppressed TJP1;

Figure 13 has eliminated RPMI 8226 cells replying ten thousand jade-like stone G1 phase retention effects for the TJP1 expression inhibiting;

Figure 14 produces for the TJP1 expression inhibiting causes resistance, makes ten thousand jade-like stones lose the restraining effect of on cell proliferation.

Embodiment

Embodiment one:

The correlation analysis of TJP1 genetic expression and tumour patient chemotherapy effect.

By high-throughput, extensive RNA interference library screening operation that we set up, we filter out TJP1 is the potential chemotherapy medicament sensitive gene.If TJP1 has determined the susceptibility of tumour patient to chemotherapeutics, TJP1 expression male patient just should respond to chemotherapeutics so, otherwise just shows as chemotherapeutics insensitive.For this reason, we have carried out the gene mapping expression analysis, with the result of treatment that determines whether TJP1 genetic expression and patient significant correlation are arranged.The gene expression data base that we adopt comes from U.S. Multiple Myeloma Genomics Portal (http://www.broad.mit.edu/mmgp/pages/publicPortalHome.jsf), wherein the Millennium database includes the gene expression chip data of 264 routine myelomatosis patients after ten thousand jade-like stones or dexamethasone treatment, and each patient is to the effect of pharmacological agent.When analyzing, we at first classify to patient, and being divided into is two big classes, and promptly 126 routine invalid group, 113 examples effectively or the effective group (being referred to as effective group) of part.Other has 25 routine patients' gene chip data noise excessive, does not add statistical study.Whether the method that we use two sample t-test relatively 34 genes and uses BUM to obtain wrong discovery rate (FDR) at invalid group with effectively there were significant differences in the group expresses.By analysis, we find that chemotherapeutics has result of treatment to the patient of TJP1 genetic expression, to the patient who does not express TJP1 then invalid (with reference to figure 1, Fig. 2, wherein NR: invalid group, R: effective group).

Embodiment two:

Quantitatively determine the expression of TJP1 gene in tumour cell.

Quantitative PCR is used to determine the expression level of TJP1 in tumour cell.Adopt QIAGENRNeasy test kit (catalog number 74104) to prepare total RNA from tumour cell, preparation process is carried out in strict accordance with product description.Wherein, the homogenate of tissue and cell adopts the QIAshredder (catalog number 79654) of QIAGEN to carry out.

Use prepared RNA concentration and the quality of spectrophotometry, getting 1 μ g RNA, to carry out eDNA synthetic.CDNA synthetic agent box adopts Invitrogen Corporation's Super Script IIIFirst-Strand Synthesis Super Mix (catalog number 18080-400) to carry out, and synthesis step is carried out in strict accordance with product description.

When carrying out the quantitative PCR reaction, the synthetic cDNA of institute is done 5 times of dilutions with the nuclease free deionized water, the cDNA that gets 1 μ l dilution is in 0.2ml

In the Optical reaction tubes, add 1 μ l TJP1TaqMan probe, 10 μ l

Gene Expression Master Mix and 8 μ l nuclease free deionized waters.The GAPDH gene is used as contrast and carries out the quantitative PCR reaction simultaneously.React among the 7900HT Fast Real-Time PCR System that is placed in Applied Biosystems and carry out.

We use the quantifying PCR method of this foundation the expression of TJP1 in 11 representative myeloma cell strains are measured, we find to have the expression of three cell strains (ARP-1, INA6, and MOLP-8) disappearance TJP1, there is a cell strain only to express extremely low TJP1 (OPM-2), all the other 7 cell strains (ANBL6, ARK, H929, KAS-6/1, RPMI 8226, MM1.s, and U266) there is TJP1 in various degree to express (as shown in Figure 3).

Embodiment three:

TJP1 genetic expression and tumour cell chemotherapeutics medial lethal dose (IC50) significant correlation.

We carry out IC50 relatively to representative MOLP-8 (no TJP1 expresses) and MM1.s (TJP1 high expression level) clone, the particular content of experiment is suddenly: WST-1 (available from RocheDiagnostics company) is used to determine the influence of adriamycin and Platinol cisplatin on cell proliferation, thereby obtains the IC50 data.Cell is inoculated in 96 well culture plates with the density of 2x104/ culture hole, is exposed in the adriamycin and Platinol cisplatin of different concns the control cells solvent treatment that does not contain medicine after 24 hours respectively.After 48 hours, cell proliferation is measured with WST-1, and measuring method is pressed the strict execution of product description.Found that MM1.s cell that TJP1 expresses all significantly is lower than MOLP-8 (being respectively 112nM and 70.2nM) (with reference to figure 4, Fig. 5) to the IC50 (being respectively 38.2nM and 13nM) of adriamycin and Platinol cisplatin, proves that the expression of TJP1 and tumour cell are closely related to the susceptibility of medicine.

Embodiment four:

Being expressed in the myelomatosis ten thousand jade-like stone drug-resistant cell strains of TJP1 lost.

Because ten thousand jade-like stones are main chemotherapeutics of myelomatosis patient, and our gene mapping analysis shows that also the result of treatment of the expression of TJP1 and ten thousand jade-like stones is closely related.Therefore, our decision take to carry out next step experiment to obtain more direct evidence.We express male ten thousand jade-like stone responsive type myeloma cell strain RPMI 8226 to TJP1 and carry out medicament screening experiment, promptly under the long-term cultivation of low concentration ten thousand jade-like stones (5nM) (March), filter out ten thousand jade-like stones are produced chemical sproof novel cell strain RPMI 8226/BR, this cell strain shows the active obvious resistance of ten thousand jade-like stone neoplasm growths, and can continue survival (with reference to figure 6, Fig. 7) in the presence of ten thousand jade-like stones of higher concentration (50nM).Afterwards, the expression of TJP1 that we have used quantitative PCR detection found that being expressed among the RPMI 8226/BR of TJP1 lose fully, means that strongly TJP1 may produce resistance play an important role on (Fig. 8) at RPMI 8226/BR to ten thousand jade-like stones.

Embodiment five:

The effect of TJP1 in chemotherapy sensitizing.

Though above-mentioned experimental result is encouraging, we still need direct evidence to prove that TJP1 can play the effect of chemotherapy sensitizing really.Therefore, we have at first carried out the chemotherapy medicament sensitive determination test to the Hela cell (HeLa/TJP1-knockdown cell) of TJP1 expression silencing.Zorubicin, cis-platinum and methotrexate are respectively applied for handles the HeLa/TJP1-knockdown cell, found that with non-target control group and compare, the HeLa/TJP1-knockdown cell has shown the resistance (with reference to figure 9, Figure 10, Figure 11) to said medicine, shows novel multiple medicines thing sensitivity genes of TJP1 representative.

Embodiment six:

Because RNA may exist non-special false positive and the effect of missing the target (off-targeteffect) in disturbing, and in order further to verify the effect of TJP1 in chemotherapy sensitizing, we have selected for use other 5 new siRNA sequences (to be called respectively: shT1, shT2, shT3, shT4, and shT5) slow virus the TJP1 among the RPMI 8226 expressed carried out the gene silencing test.Above-mentioned slow virus is available from U.S. Sigma company.RPMI 8226 cells are infected slow virus respectively, and control cells infects non-special target virus.Infect after 24 hours, 1500 rev/mins of eccentric cells abandon old nutrient solution, change fresh medium into, cultivate 10 days with the nutrient solution that contains 1 μ g/ml tetracycline after 24 hours, change one time nutrient solution in wherein per two days.Use quantitative PCR detection TJP1 expression of gene at last, found that one of them siRNA sequence (shT2) can effectively suppress＞expression (with reference to Figure 12) of 75%TJP1.

Then, we analyzed the cell cycle of non-target (shN), shT2 and shT4 cell.Cell fixedly spends the night with 70% ethanol, after phosphoric acid buffer cleans, in 37C processing 20 minutes, carries out cell cycle analysis then on cell streaming instrument in the propidium iodide (Propidium Iodide) of 50 μ g/ml and RNaseA (20 μ g/ml).Found that shN and sh4 (no TJP1 expression inhibiting) cell have similar cell cycle collection of illustrative plates after ten thousand jade-like stones and cisplatin treated, its G1 phase cell obviously increases; The sh2 cell then shows the negligible G1 phase and changes (with reference to Figure 13).We go back the increment of pair cell simultaneously and have carried out the WST-1 detection, and consistent with the cell cycle data, sh2 cell proliferation is not subjected to the obvious influence (with reference to Figure 14) of chemotherapeutics.The above results proves that strongly the expression inhibiting of TJP1 can cause the resistance of cell to chemotherapeutics.

Should be pointed out that for the present invention also to have the embodiment of multiple conversion and remodeling, be not limited to the specific embodiment of above-mentioned embodiment through proving absolutely.The foregoing description is as just explanation of the present invention, rather than restriction.In a word, protection scope of the present invention should comprise those conspicuous to those skilled in the art conversion or substitute and remodeling.

Claims

1. tumour chemotherapy medicament sensitive gene, this gene is the TJP1 gene.

2. one kind has gene and the proteins encoded thereof that strengthens the chemotherapeutics drug effect to tumour cell, and this gene is the TJP1 gene.

3. method of predicting the chemotherapy of tumors effect is characterized in that: described method comprises that with the TJP1 gene as the target that detects, when having TJP1 to express in the patient body, patient has susceptibility to chemotherapeutics; When in the patient body during TJP1 expression deletion, patient table reveals the resistance to chemotherapeutics.

4. method that improves the chemotherapy of tumors susceptibility is characterized in that: described method comprises the TJP1 gene as target, induces and/or improves natural TJP1 expression of gene, to improve the chemotherapy susceptibility.

5. the conduct in prediction chemotherapy of tumors effect of the described TJP1 gene of claim 1 detects the application of target.

6. the described TJP1 gene of claim 1 is as the application of the target that improves the chemotherapy of tumors susceptibility.

7. the application of the described TJP1 gene of claim 1 in the preparation anti-tumor medicine.