阅读说明：本技术 生物标志物及其应用 (Biomarkers and uses thereof ) 是由 高山 蒋思远 于 2020-03-16 设计创作，主要内容包括：本发明涉及一种生物标志物及其应用。该生物标志物能够用于评估IGF1R抑制剂对IGF1R的抑制作用,生物标志物包括HUNK,IGF1R抑制剂包括BMS。上述生物标志物能够用于有效评估BMS抑制剂对IGF1R抑制作用。(The invention relates to a biomarker and application thereof. The biomarkers can be used to assess the inhibitory effect of IGF1R inhibitors on IGF1R, the biomarkers including HUNK and IGF1R inhibitors including BMS. The biomarkers can be used for effectively evaluating the inhibition effect of the BMS inhibitor on IGF 1R.)

1. A biomarker capable of being used to assess the inhibitory effect of an IGF1R inhibitor on IGF1R, said biomarker comprising HUNK, and said IGF1R inhibitor comprising BMS-754807.

2. The biomarker of claim 1, further comprising at least one of AGAP3 and IGF 2R.

3. The biomarker of any of claims 1 to 2, further comprising at least one of JIP4, ARF6 and p-MKK4, MKK4, p-JNK, p-c-Jun and c-Jun.

4. The biomarker of any of claims 1 to 2, wherein the IGF1R inhibitor further comprises OSI-906.

5. Use of the biomarker of any of claims 1 to 4 in the preparation of a test kit for detecting the anti-tumor effect of the IGF1R inhibitor or a test device for detecting the anti-tumor effect of the IGF1R inhibitor.

6. The use of claim 5, wherein the tumor is colon cancer.

7. A method of increasing the inhibitory effect of an IGF1R inhibitor on IGF1R, comprising the steps of: knocking out a biomarker gene of an organism to increase the inhibitory effect of an IGF1R inhibitor on IGF1R, the biomarker including HUNK, the IGF1R inhibitor including BMS-754807;

or, comprising the following steps: administering to the body an inhibitor that inhibits the gene expression of a biomarker comprising HUNK, and an IGF1R inhibitor comprising BMS-754807, to increase the inhibitory effect of the IGF1R inhibitor on IGF 1R.

8. An antitumor pharmaceutical composition, comprising a HUNK inhibitor and an IGF1R inhibitor, wherein the IGF1R inhibitor comprises BMS-754807.

9. The anti-neoplastic pharmaceutical composition according to claim 8, wherein said HUNK inhibitor comprises at least one of lapatinib and staurosporine.

10. The antineoplastic pharmaceutical composition of claim 8, wherein said IGF1R inhibitor further comprises OSI-906;

and/or the tumor is colon cancer.

11. An assay kit for detecting the anti-tumor effect of an IGF1R inhibitor, comprising a detector that specifically binds to a biomarker comprising HUNK, said IGF1R inhibitor comprising BMS-754807.

12. The test kit for detecting the anti-tumor effect of an IGF1R inhibitor according to claim 11, characterized in that the detector specifically binding to a biomarker comprises a primer pair for amplifying the HUNK gene.

13. The detection kit for detecting the antitumor effect of an IGF1R inhibitor according to claim 12, wherein the base sequence of the primer pair for amplifying the HUNK gene is represented by SEQ ID No.1 to SEQ ID No. 2.

Technical Field

The invention relates to the technical field of biology, in particular to a biomarker and application thereof.

Background

Insulin-like growth factors (IGFs) are a class of cytokines with a wide range of biological functions. IGFs include both IGF-1 and IGF-2 growth factors, which are evolutionarily conserved active peptides with similar structures to insulin. Among them, in most cells and tissues, IGF-1 promotes cell proliferation, differentiation and survival, affecting the metabolism of insulin. The insulin-like growth factor 1receptor (IGF-1receptor, IGF1R) mediates the action of IGF-1.

IGF1R is a member of the Receptor Tyrosine Kinases (RTKs) family, including insulin, Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF), and platelet-derived growth factor (PDGF) receptors, among others IGF1R is a heterotetrameric transmembrane protein, consisting of two α -subunits and two β -subunits, with 3 disulfide bonds between the α -and β -subunits, and IGF1R inhibitor has inhibitory effects on IGF 1R. among others, it would be advantageous to screen for suitable IGF1R inhibitors by evaluating the inhibitory effects of IGF1R inhibitors on IGF 1R. however, the prior art lacks a means for effectively evaluating the inhibitory effects of IGF1R inhibitors, and in particular, the inhibition effects of BMS inhibitors are difficult to evaluate, and do not meet the actual need.

Disclosure of Invention

Based on this, there is a need for providing a biomarker which can be used for effectively evaluating the inhibition effect of the BMS inhibitor on IGF1R and the use thereof.

A biomarker capable of being used to assess the inhibitory effect of an IGF1R inhibitor on IGF1R, said biomarker comprising HUNK, said IGF1R inhibitor comprising BMS.

The research carries out a great deal of research on IGF1R inhibitor, and unexpectedly discovers that the expression level of HUNK gene influences the effect of BMS-754807 inhibitor on IGF1R inhibitor, so that HUNK can be used as a biomarker to effectively evaluate the inhibition effect of BMS-754807 inhibitor on IGF 1R.

In one embodiment, the biomarker further comprises at least one of AGAP3 and IGF 2R.

In one embodiment, the biomarker further comprises at least one of JIP4, ARF6, p-MKK4, MKK4, p-JNK, p-c-Jun, and c-Jun.

In one embodiment, the IGF1R inhibitor further comprises OSI-906.

The application of the biomarker in preparing a detection kit for detecting the anti-tumor effect of the IGF1R inhibitor or a detection device for detecting the anti-tumor effect of the IGF1R inhibitor.

In one embodiment, the tumor is colon cancer.

A method of increasing the inhibitory effect of an IGF1R inhibitor on IGF1R, comprising the steps of: knocking out a biomarker gene of an organism to increase the inhibitory effect of an IGF1R inhibitor on IGF1R, the biomarker including HUNK, the IGF1R inhibitor including BMS-754807;

or, comprising the following steps: administering to the body an inhibitor that inhibits the gene expression of a biomarker comprising HUNK, and an IGF1R inhibitor comprising BMS-754807, to increase the inhibitory effect of the IGF1R inhibitor on IGF 1R.

An antitumor pharmaceutical composition comprises HUNK inhibitor and IGF1R inhibitor, wherein the IGF1R inhibitor comprises BMS-754807.

In one embodiment, the HUNK inhibitor comprises at least one of lapatinib and staurosporine.

In one embodiment, the IGF1R inhibitor further comprises OSI-906;

and/or the tumor is colon cancer.

An assay kit for detecting an anti-tumor effect of an IGF1R inhibitor, comprising a detector that specifically binds to a biomarker comprising HUNK, said IGF1R inhibitor comprising BMS-754807.

In one embodiment, the detector that specifically binds to the biomarker comprises a primer pair that amplifies the HUNK gene.

In one embodiment, the base sequence of the primer pair for amplifying the HUNK gene is shown as SEQ ID No. 1-SEQ ID No. 2.

Drawings

FIG. 1 is a graph comparing the relative cell proliferation of wild-type SW480 human colon cancer cells and HUNK gene knocked out SW480 human colon cancer cells at different concentrations of OSI-906 treatment in example 1;

FIG. 2 is a graph comparing the relative cell proliferation of gene-knocked-down placebo SW480 human colon cancer cells, wild type SW480 human colon cancer cells after their first HUNK target knockdown, wild type SW480 human colon cancer cells after their second HUNK target knockdown at different concentrations of OSI-906 treatment in example 1;

FIG. 3 is a graph comparing the relative cell proliferation of HUNK overexpressing HT-29 human colon cancer cells, a placebo HT-29 human colon cancer cells overexpressing HUNK, under different concentrations of OSI-906 treatment in example 1;

FIG. 4 is a graph comparing the relative cell proliferation amounts of wild-type SW480 human colon cancer cells and HUNK gene-knocked out SW480 human colon cancer cells under different concentrations of BMS-754807 treatment in example 1;

FIG. 5 is a graph comparing the relative cell proliferation of the gene-knocked-down blank SW480 human colon cancer cells, wild type SW480 human colon cancer cells after the first HUNK target knockdown, wild type SW480 human colon cancer cells after the second HUNK target knockdown, treated with BMS-754807 at different concentrations in example 1;

FIG. 6 is a graph comparing the relative cell proliferation of HUNK-overexpressing HT-29 human colon cancer cells, a blank control of HUNK-overexpressing HT-29 human colon cancer cells, and HUNK-overexpressing HT-29 human colon cancer cells, treated with BMS-754807 at different concentrations in example 1;

FIG. 7 is a photograph of each group of cells cultured in the 6-well plate in example 2;

FIG. 8 is a graph showing a comparison of the number of clone formation of each group of cells cultured in the 6-well plate in example 2;

FIG. 9 is a photograph of each group of cells cultured in the 96-well plate in example 2;

FIG. 10 is a graph comparing the ratio of the relative three-dimensional spherical area of each group of cells cultured in the 96-well plate in example 2;

FIG. 11 is a graph comparing the change of tumor volume with the culture time after gavage of various groups of mice;

FIG. 12 is a photograph of tumors after intragastric administration in each group of mice;

FIG. 13 is a graph comparing tumor volumes of groups of mice cultured for 10 days by gavage;

FIG. 14 is a graph comparing the gene expression amounts of HUNK and IGF2R in wild-type SW480 human colon cancer cells and HUNK gene-knocked out SW480 human colon cancer cells in example 4;

FIG. 15 is the mRNA expression assay of IGF2R after HUNK knockdown in SW480 cell line in example 4;

FIG. 16 is a graph showing a comparison of the gene expression levels of HUNK and IGF2R in HT-29 human colon cancer cells, HUNK overexpressed HT-29 human colon cancer cells in example 4;

FIG. 17 is a graph comparing the protein expression amounts of HUNK, IGF2R and GADPH of wild-type SW480 human colon cancer cell and HUNK gene-knocked out SW480 human colon cancer cell in example 4;

FIG. 18 shows the results of the detection of the protein expression of IGF2R after HUNK knockdown in SW480 cell line in example 4;

FIG. 19 is a graph comparing the protein expression amounts of HUNK, IGF2R and GADPH of wild-type SW480 human colon cancer cell and HUNK gene-knocked out SW480 human colon cancer cell in example 4;

fig. 20 is a graph comparing the relative cell proliferation amounts of sgRNA and sgIGF2R in example 5;

fig. 21 is a graph comparing the relative cell proliferation amounts of shNC, shIGF2R1, shIGF2R2 in example 5;

FIG. 22 is a graph showing a comparison of relative cell proliferation amounts of NEO shNC, OE HUNK shIGF2R1, and OE HUNKshIGF2R2 in example 5;

FIG. 23 is a graph comparing the relative cell proliferation amounts of WT sgRNAs, KO sgIGF2R1, and KO sgIGF2R2 in example 5;

FIGS. 24 to 27 are graphs showing comparison of expression levels of HUNK, AGAP3, MKK4, p-MKK4, ARF6, JNK, p-JNK, c-JUN, p-c-JUN, JIP4, IGF2R and GADPH in example 6;

FIG. 28 is a graph showing the protein expression levels of HER2, HUNK, AGAP3, MKK4, p-MKK4, ARF6, JNK, p-JNK, c-JUN, p-c-JUN, JIP4 and GADPH in example 7;

FIG. 29 is a graph comparing the cellular activities of L apatinib group and OSI-906 (10. mu.M) -L apatinib group in example 7;

FIG. 30 is a graph comparing the tumor volume after gavage with the culture time in the mice of each group in example 7;

FIG. 31 is a photograph of tumors after intragastric administration in each group of mice in example 7;

FIG. 32 is a graph comparing tumor volumes of groups of mice cultured by gavage for 10 days in example 7;

FIG. 33 is a Western blot of the expression level of IGF2R after knock-down of c-Jun protein by SW480 cells in example 8;

FIG. 34 is a Western blot of the expression level of IGF2R after SW480 cells overexpress c-Jun protein in example 8;

FIG. 35 is a Western blot of the expression level of IGF2R after simultaneous knock-down of c-Jun in SW480 cell line overexpressing HUNK protein in example 8;

FIG. 36 is a Western blot of the expression level of IGF2R after JNK inhibitor application of SW480 cell line overexpressing HUNK protein in example 8;

FIG. 37 is a Western blot of expression levels of both JNK phosphate and JNK, phosphorylated c-Jun and c-Jun, and IGF2R in the SW480 cell line overexpressing HUNK protein in example 8 after knocking down MKK4 simultaneously;

FIG. 38 shows the cell activity of SW480 cell line knocked-down IGF2R in example 8 using BMS-754807 inhibitor;

fig. 39 is a graph of the cellular activities of SW480 cell line overexpressing IGF2R using BMS inhibitors in example 8.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The biomarker of one embodiment can be used for evaluating the inhibition effect of an IGF1R inhibitor on IGF1R, so that the biomarker can be used for preparing a test kit for detecting the antitumor effect of the IGF1R inhibitor or a test device for detecting the antitumor effect of the IGF1R inhibitor. The tumor may be, for example, colon cancer. Specifically, biomarkers include HUNK. IGF1R inhibitors include BMS.

Hormone up-regulated tumor-associated kinase (HUNK) was originally named Bstk1, also known as VMR tumor family metastasis associated protein kinase (VMR tumor kinase in Mak-V). The HUNK gene is located on chromosome 21, is 80kDa in length, and is a SNF 1-related AMPK family serine/threonine kinase. The research carries out a great deal of research on IGF1R inhibitor, and unexpectedly discovers that the expression level of HUNK gene influences the effect of BMS inhibitor on IGF1R inhibitor, so that HUNK can be used as biomarker to effectively evaluate the inhibition effect of BMS inhibitor on IGF 1R.

In one embodiment, the BMS inhibitor is a BMS-754807 inhibitor. The BMS inhibitor is not limited to BMS-754807 inhibitor, and may be other BMS inhibitors that inhibit IGF 1R.

In one embodiment, the biomarker further comprises AGAP 3. AGAP3 is a GTPase activating protein of the ADP ribosylating factor family with a GTP hydrolase domain. The research finds that HUNK can up-regulate the expression of AGAP3, so that the inhibition effect of an IGF1R inhibitor on IGF1R can be more accurately evaluated by taking AGAP3 and HUNK as biomarkers, and the specificity is higher.

In one embodiment, the biomarker further comprises at least one of JIP4 and ARF 6. JIP4 is a JNK scaffold protein. ARF6 is a GTP-binding protein with ADP-ribosyltransferase activity. The present study found that HUNK can up-regulate the expression of jis 4 and ARF6, and therefore, by using jis 4, ARF6 and HUNK as biomarkers, the inhibitory effect of IGF1R inhibitors on IGF1R can be more accurately evaluated, and the specificity is high.

In one embodiment, the biomarkers further comprise at least one of p-MKK4, MKK4, p-JNK, p-c-Jun, and IGF 2R. p-MKK4 is a phosphorylated mitogen-activated protein kinase 4. ARF6 is ADP-ribosyltransferase 6. MKK4 is a mitogen-activated protein kinase 4. p-JNK is a phosphorylated c-Jun amino terminal kinase. JNK is c-Jun amino terminal kinase. p-c-Jun is phosphorylated c-Jun. c-Jun is a transcription regulator and belongs to a leucine zipper family member. IGF2R is an insulin-like growth factor 2receptor (IGF 2receptor, IGF2R) that mediates the actions of IGF-2. The research discovers that HUNK can influence the expression of p-MKK4, MKK4, p-JNK, p-c-Jun, c-Jun and IGF2R, so that the inhibition effect of the IGF1R inhibitor on IGF1R can be more accurately evaluated by taking p-MKK4, MKK4, p-JNK, p-c-Jun, IGF2R and HUNK as biomarkers, and the specificity is higher.

In one embodiment, the IGF1R inhibitor further comprises OSI-906. The present study found that HUNK as a biomarker was able to assess not only the inhibitory effect of BMS, but also the inhibitory effect of other IGF1R inhibition, such as OSI-906. It should be noted that the IGF1R inhibitor is not limited to the above-mentioned IGF1R inhibitor, but may be other IGF1R inhibitors commonly used in the art.

The research carries out a great deal of research on IGF1R inhibitor, and unexpected discovery shows that the expression level of HUNK gene influences the effect of BMS inhibitor on IGF1R inhibitor, so that HUNK can be used as a biomarker to effectively evaluate the inhibition effect of BMS inhibitor on IGF1R, and the HUNK gene can be used as a combined target of IGF1R inhibitor.

One embodiment of a method of increasing the inhibitory effect of an IGF1R inhibitor on IGF1R, comprises the steps of: knocking out organism biomarker genes to improve the inhibition effect of IGF1R inhibitor on IGF1R, wherein the biomarkers comprise HUNK and the IGF1R inhibitor comprises BMS.

In one embodiment, the IGF1R inhibitor further comprises OSI-906. It should be noted that the IGF1R inhibitor is not limited to the above-mentioned IGF1R inhibitor, but may be other IGF1R inhibitors commonly used in the art.

The research shows that the HUNK gene can improve the impedance effect of cells on IGF1R inhibitors. Therefore, the inhibition effect of the IGF1R inhibitor on IGF1R can be improved by knocking out the HUNK gene of the body.

Another embodiment of a method of increasing the inhibitory effect of an IGF1R inhibitor on IGF1R, comprising the steps of: administering to the body an inhibitor that inhibits the expression of the biomarker gene to increase the inhibitory effect of the IGF1R inhibitor on IGF 1R. Biomarkers include HUNK. IGF1R inhibitors include BMS.

In one embodiment, the IGF1R inhibitor further comprises OSI-906. It should be noted that the IGF1R inhibitor is not limited to the above-mentioned IGF1R inhibitor, but may be other IGF1R inhibitors commonly used in the art.

The research shows that the HUNK gene can improve the impedance effect of cells on IGF1R inhibitors. Therefore, the inhibition of IGF1R by IGF1R inhibitor is increased by administering to the body an inhibitor that inhibits the expression of the HUNK gene to inhibit the expression of the HUNK gene.

In addition, the anti-tumor pharmaceutical composition of the embodiment has a better anti-tumor effect. Specifically, the antitumor drugs include HUNK inhibitors and IGF1R inhibitors. IGF1R inhibitors include BMS.

In one embodiment, the HUNK inhibitor comprises at least one of lapatinib and staurosporine. The HUNK inhibitor can be matched with an IGF1R inhibitor to improve the anti-tumor effect of the anti-tumor pharmaceutical composition. It should be noted that the HUNK inhibitors are not limited to the above-mentioned HUNK inhibitors, but may be other HUNK inhibitors commonly used in the art.

In one embodiment, the IGF1R inhibitor further comprises OSI-906. HUNK inhibitor can be combined with OSI-906 to improve antitumor effect of the antitumor pharmaceutical composition. It should be noted that the IGF1R inhibitor is not limited to the above-mentioned IGF1R inhibitor, but may be other IGF1R inhibitors commonly used in the art.

In one embodiment, the molar ratio of the HUNK inhibitor to IGF1R inhibitor is 1: 1-1: 100.

in one embodiment, the tumor is colon cancer.

According to the research, a large number of researches are carried out on an IGF1R inhibitor, and unexpected findings are found that the expression quantity of the HUNK gene influences the effect of the BMS inhibitor on an IGF1R inhibitor, so that the HUNK can be used as a biomarker to effectively evaluate the inhibition effect of the BMS inhibitor on IGF1R, and the HUNK gene can be used as a combined target spot of the IGF1R inhibitor, so that the anti-tumor pharmaceutical composition with excellent anti-tumor effect is obtained by matching the HUNK inhibitor and the IGF1R inhibitor.

One embodiment of the test kit for detecting an anti-tumor effect of an IGF1R inhibitor comprises a detector that specifically binds to a biomarker. Biomarkers include HUNK. IGF1R inhibitors include BMS.

In one embodiment, the detector that specifically binds to the biomarker is a primer capable of amplifying the biomarker or a probe capable of detecting the biomarker. The content of the biomarker is quantitatively or qualitatively determined by amplifying the primer of the biomarker or the probe for detecting the biomarker.

In one embodiment, the detector that specifically binds to the biomarker comprises a primer pair for PCR amplification of the HUNK gene. It should be noted that the detection object specifically binding to the biomarker is not limited to a primer pair including PCR amplification of the HUNK gene, and may include a probe for detecting the HUNK gene.

In one embodiment, the base sequence of the primer pair for PCR amplification of HUNK gene is shown in SEQ ID No. 1-SEQ ID No. 2. Specifically, the sequence shown as SEQ ID No.1 is 5'-CCCGCAGATACATCCGACAG-3'. The sequence shown as SEQ ID No.2 is 5'-TTTTGGGGCCGTATTTCTTCC-3'.

In one embodiment, the BMS inhibitor is a BMS-754807 inhibitor. The BMS inhibitor is not limited to the BMS-754807 inhibitor, and may be other BMS inhibitors.

In one embodiment, the biomarker further comprises AGAP 3. The research finds that HUNK can up-regulate the expression of AGAP3, so that the inhibition effect of an IGF1R inhibitor on IGF1R can be more accurately evaluated by taking AGAP3 and HUNK as biomarkers, and the specificity is higher. Further, the detector specifically binding to the biomarker further comprises a primer pair for PCR amplification of the AGAP3 gene.

In one embodiment, the biomarker further comprises at least one of JIP4 and ARF 6. The present study found that HUNK can up-regulate the expression of jis 4 and ARF6, and therefore, by using jis 4, ARF6 and HUNK as biomarkers, the inhibitory effect of IGF1R inhibitors on IGF1R can be more accurately evaluated, and the specificity is high. Further, the detector that specifically binds to the biomarker further includes at least one of a primer pair for PCR amplification of the jis 4 gene and a primer pair for PCR amplification of the jis 4 gene.

In one embodiment, the biomarkers further comprise at least one of p-MKK4, MKK4, p-JNK, p-c-Jun, and IGF 2R. The research discovers that HUNK can influence the expression of p-MKK4, MKK4, p-JNK, p-c-Jun, c-Jun and IGF2R, so that the inhibition effect of the IGF1R inhibitor on IGF1R can be more accurately evaluated by taking p-MKK4, MKK4, p-JNK, p-c-Jun, IGF2R and HUNK as biomarkers, and the specificity is higher. Further, the detector specifically binding to the biomarker further comprises at least one of a primer pair for PCR amplification of the p-MKK4 gene, a primer pair for PCR amplification of the MKK4 gene, a primer pair for PCR amplification of the p-JNK gene, a primer pair for PCR amplification of the p-c-Jun gene, a primer pair for PCR amplification of the c-Jun gene and a primer pair for PCR amplification of the IGF2R gene.

In one embodiment, the IGF1R inhibitor further comprises OSI-906. The present study found that HUNK as a biomarker was able to assess not only the inhibitory effect of BMS, but also the inhibitory effect of other IGF1R inhibition, such as OSI-906. It should be noted that the IGF1R inhibitor is not limited to the above-mentioned IGF1R inhibitor, but may be other IGF1R inhibitors commonly used in the art.

In one embodiment, the kit for detecting the anti-tumor effect of the IGF1R inhibitor further comprises DNA polymerase and reagents commonly used in PCR reaction of biomarkers, and can be matched with a detector specifically binding to the biomarkers to realize the detection of the biomarkers.

Wherein the DNA polymerase is at least one selected from T4 DNA polymerase, Klenow enzyme and DNA polymerase I. Reagents include, for example, buffers, dNTPs (deoxyribonucleoside triphosphates including dATP, dGTP, dTTP, dCTP), fluorescent dyes, and stabilizers.

In one embodiment, the test kit for detecting the antitumor effect of the IGF1R inhibitor further comprises a reagent for detecting an internal reference gene. For example, a PCR amplification primer pair for the reference gene may be included. The content of the reference gene is detected by a reagent for detecting the reference gene, and the content of the biological standard is calculated by taking the content as a reference, so that the experimental error existing in the detection process of the biomarker is corrected, and the accuracy of the experimental result is ensured. Specifically, the reference gene may be GAPDH, for example.

The detection kit can evaluate the anti-tumor effect of the IGF1R inhibitor by detecting the biomarkers, and the obtained result has high sensitivity and high specificity, so that the screening of the IGF1R inhibitor is facilitated, and the anti-tumor research has important clinical application value.

The following are specific examples.