LU505210B1

LU505210B1 - Marker probe for predicting sensitivity of chemotherapeutic drug for colorectal cancer and application

Info

Publication number: LU505210B1
Application number: LU505210A
Authority: LU
Inventors: Bianqin Guo
Original assignee: Chongqing Univ Cancer Hospital
Priority date: 2023-09-28
Filing date: 2023-09-28
Publication date: 2024-03-28

Abstract

The present invention belongs to the technical field of gene detection, and particularly discloses a marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC, including a PHLPP probe for detecting PHLPP1 and PHLPP2, where the nucleotide sequence of the probe for detecting PHLPP1 is one or a combination of SEQ ID NO. 1 to SEQ ID NO. 8, and the nucleotide sequence of the probe for detecting PHLPP2 is one or a combination of SEQ ID NO. 9 to SEQ ID NO. 16. The present invention further discloses a method for detecting the marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC and an application of the probe and the detection method in preparation of a detection kit.

Description

DESCRIPTION LU505210

MARKER PROBE FOR PREDICTING SENSITIVITY OF CHEMOTHERAPEUTIC

DRUG FOR COLORECTAL CANCER AND APPLICATION

TECHNICAL FIELD

[01] The present invention belongs to the technical field of gene detection, and particularly relates to a marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC and an application thereof.

BACKGROUND ART

[02] Colorectal Cancer (CRC) is one of the major malignant tumors threatening human health. Global cancer statistics show that in 2020, the incidence rate of CRC accounted for 10.0% of all cancers in the world, and the death rate thereof accounted for 9.4% of the total cancer induced deaths. In China, CRC ranks only second to lung cancer in the incidence rate, becoming the second largest cancer. Epidemiological data have revealed that in recent years, both the incidence rate and the death rate of CRC in

China have been on the rise. Clinical manifestations of CRC mainly include change of bowl evacuation habit, abdominal pain, anemia, and enterorrhagid. However, most patients have no obvious symptoms in early stage, which causes CRC patients to be newly diagnosed in middle and advanced stages.

[03] At present, the treatment method for the CRC patients in the middle and advanced stages where tumors cannot be surgically excised and the CRC patients who are not suitable for immunotherapy is still based on chemotherapy.

[04] The intestinal cancer guidebook Edition II (V2) 2021 newly updated by National

Comprehensive Cancer Network (NCCN) still recommends use of FOLFOX (Oxaliplatin+calcium folinate+5-Fluorouracil (5-FU) or CAPEOX (Oxaliplatin+Capecitabine) as a major auxiliary treatment means.

[05] In recent years, the liquid biopsy technology represented by circulating tumor cell (CTC) typing detection has become an emerging hotspot in the field of tumor research, which has better application prospects in the fields of early diagnosis, staging, curative effect monitoring, and prediction and evaluation of tumor patients. CRC of most CRC patients in the middle and advanced stages will relapse after the patients receive chemotherapy, with the five-year survival rate lower than 15%. Chemotherapy resistance is the primary cause resulting in unfavorable prognosis of the patients and relapse of the disease.

[06] Existing technical means mainly include detecting some genes related to tumor drugs (for example, TS genes, MTHFR genes, and the like) through tissue biopsy or blood samples to predict the sensitivity of the patients to the chemotherapeutic drugs.

However, the sampling difficulty of tissue biopsy is great and sampling cannot be repeated, which, therefore, is not conducive to dynamically evaluating the drug resistance. Drug related gene detection through peripheral plasma or serum mainly aims to detect fragmented DNAS released after tumor cell necrosis, which cannot truly reflect the actual condition of the tumors.

[07] There are two subtypes (PHLPP1 and PHLPP2) in the PHLPP phosphatase family, which belong to a PPM superfamily of serine/threonine protein phosphatase.

Upon initial discovery of PHLPP as the protein phosphatase of AKT, many researchers verify that the PHLPP inhibits occurrence and development of CRC by inhibiting various oncogenic signal paths. It is found in the previous research (Cell Death and Disease (2021)12:960; https://doi.0rg/10.1038/s41419-021-04251-0) by the applicant that down-regulation of the PHLPP mainly causes chemotherapy resistance of CRC, and the mechanism by which the PHLPP induces drug resistance of CRC is proved, indicating that the PHLPP can serve as a marker for predicting the sensitivity of a chemotherapeutic drug for CRC. However, at present, there have been no feasible kits and corresponding detection means clinically for detecting the expression of mRNA of the PHLPP.

SUMMARY LU505210

[08] An objective of the present invention is to provide a marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC and an application thereof, so as to solve the above technical problems.

[09] The first objective of the present invention is to provide a marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC. The probe includes a

PHLPP probe for detecting PHLPP1 and PHLPP2, where the nucleotide sequence of the probe for detecting PHLPP1 is one or a combination of SEQ ID NO. 1 to SEQ ID NO. 8, and the nucleotide sequence of the probe for detecting PHLPP2 is one or a combination of SEQ ID NO. 9 to SEQ ID NO. 16.

[10] Preferably, the marker probe further includes epitheliated peripheral blood CTC specific capture probes EpCAM, CK8, CK18, and CK19, with the nucleotide sequences respectively expressed as SEQ ID NO. 17-SEQ ID NO. 22, SEQ ID NO. 23-SEQ ID NO. 28, SEQ ID NO. 29-SEQ ID NO. 34, and SEQ ID NO. 35-SEQ ID NO. 40; further includes interstitial peripheral blood CTC specific capture probes Vimentin and Twist, with the nucleotide sequences respectively expressed as SEQ ID NO. 41-SEQ ID NO. 46, and SEQ ID NO. 47-SEQ ID NO. 52; and further includes a leucocyte phenotypic specific capture probe, with the nucleotide sequences expressed as EQ ID NO. 53-SEQ

ID NO. 58.

[11] The second objective of the present invention is to provide an application of the probe in preparation of a detection kit.

[12] The third objective of the present invention is to provide a method for detectirl4/505210 the marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC, including the following steps:

[13] $1, intercepting peripheral blood CTC with a filter membrane;

[14] S2, performing typing detection on the intercepted peripheral blood CTC with three specific capture probes, where the three specific capture probes are respectively:

[15] (1) the epitheliated peripheral blood CTC specific capture probes EpCAM, CK8,

CK18, and CK19;

[16] (2) the interstitial peripheral blood CTC specific capture probes Vimentin and

Twist; and

[17] (3) the leucocyte phenotypic specific capture probe CD45.

[18] SZ’, detecting the expression condition of mRNA of PHLPP in the peripheral blood CT with a PHLPP probe.

[19] The fourth objective of the present invention is to provide an application of the detection method in preparation of the detection kit.

[20] The principle and beneficial effects of the present invention are as follows:

[21] 1, according to research results published in the early stage, the down-regulated expression of PHLPP is the primary cause of chemotherapy tolerance for CRC.

[22] 2, according to the present invention, through the designed PHLPP probes for detecting PHLPP1 and PHLPP2, the mRNA level of PHLPP of peripheral blood CTC is detected by way of nano membrane filtration in combination with the mRNA in-situ hybridization technique to predict the sensitivity of the patients to the chemotherapeutic drugs such as Oxaliplatin, 5-Fluorouracil, and Capecitabine, so as to detect and evaluate the drug resistance of the tumor cells dynamically.

[23] 3, after the peripheral blood CTC is intercepted with the filter membrane, tH&/505210 specificity of the peripheral blood CTC needs to be identified and typed with the probe. In the process of identifying and typing by the probe, CTC typing detection and PHLPP

MRNA detection can be performed synchronously without interfering with each other, so the identifying and typing detection efficiencies are high.

[24] 4, the primary cause to detect the mRNA level of the PHLPP of peripheral blood

CTC by means of the mRNA in-situ hybridization technique lies in that

[25] (1) CTC and tissue samples are highly consistent, sampling can be repeated, and the time point when drug resistance will occur on the patients can be captured timely; and

[26] (2) the mRNA in-situ hybridization technique features high specificity, high sensitivity, easy and rapid operation, and low cost.

BRIEF DESCRIPTION OF THE DRAWINGS

[27] FIG. 1 is a colonoscopic view under a fluorescence microscope, where red dotted fluorescence represents expression conditions of epithelial markers of peripheral blood

CTC: EpCAM, CK8, CK18 and CK19 genes; green dotted fluorescence represents expression conditions of interstitial markers: Vimentin and Twist genes of peripheral blood CTC; purple dotted fluorescence represents expression conditions of interstitial markers: PHLPP1 and PHLPP2 genes of peripheral blood CTC; a white signal point represents a gene expression condition of a leukocyte marker CD45; and a graph A and a graph C are composite graphs of a plurality of fluorescent channels, and a graph B and a graph D are single fluorescent channel graphs (a scientific research probe expression signal graph).

DETAILED DESCRIPTION OF THE EMBODIMENTS LU505210

[28] The present invention will be further described in detail below by means of specific implementations:

[29] 1.1 Experimental design

[30] Table 1 Experimental design 1 Epeimemaiuee [ueewes

CTC Intercept peripheral blood CTC |/ ie eme LI

Identify mRNA expression level of | Three specific capture probes

PHLPP in peripheral blood CTC | (1) the epitheliated peripheral blood with different phenotypes, and | CTC specific capture probes EpCAM, identify whether the phenotypes | CK8, CK18, and CK19; of the circulating tumor cells are | (2) the interstitial peripheral blood epitheliated or interstitial CTC specific capture probes

Vimentin and Twist; and (3) the leucocyte phenotypic specific capture probe CD45.

CTC Detect expressions of PHLPP1 | PHLPP probe identification | and PHLPP2 in the circulating tumor cells; and illustrate the expression of PHLPP1 and

PHLPP2 of PHLPP in the circulating tumor cells with different phenotypes

Detect the mRNA level of the

PHLPP in peripheral blood CTC to predict the sensitivity of a patient to the chemotherapeutic drugs such as Oxaliplatin, 5-Fluorouracil, and Capecitabine, and dynamically detect and evaluate drug resistance of the tumor cells in the treatment process, so as to observe the time points when chemotherapy resistance is generated

[31] 1.2. PHLPP probe

[32] A marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC is designed by mainly using online Primer 5 software which itself will optimize specific probe sequences by sequence comparison. The probe sequences are synthesized by

Invitrogen. Finally, through a pre-experiment of expression conditions of high, medium and low expression housekeeping genes (B2M), TBP, and TFRC), the specificity of the probe sequences is verified according to the expression conditions of the housekeeping genes, i.e., sequences listed in table 2.

[33] During a PCR experiment, to remove the probable differences of different specimens in yield, quality, and reverse transcription efficiency of RNA so as to acquire true differences of specific expressions of target genes, certain housekeeping genes are usually selected for calibration and standardization. The housekeeping genes B2M, TBP, and TFRC used in the present invention correspond to high, medium, and low expressions, respectively.

[34] Table 2 PHLPP probes LU505210

PHLPP1 SEQ ID NO. 1 gcttactettctact

SEQ ID NO. 2 gtctgtaagttgtgcatcac

SEQ ID NO. 3 tattgtctcgtaggtcaage

SEQ ID NO. 4 aagacctcgatgcagttgg

SEQ ID NO. 5 gtgtaaccatgactccatac

SEQ ID NO. 6 cgacagagaactgttggca

SEQ ID NO. 7 tccgggggtatgataaactg

SEQ ID NO. 8 gcctcactatacaacactga

PHLPP2 SEQ ID NO. 9 cgtttcatatttctcttgge

SEQ ID NO. 10 ccaaagtctcgaagctgaca

SEQ ID NO. 11 agattccattettcagg

SEQ ID NO. 12 tetgagttaaggcttgg

SEQ ID NO. 13 gttccagaaccagattgta

SEQ ID NO. 14 gatagtggctaccagtttat

SEQ ID NO. 15 tcagagtcatctgccataac

SEQ ID NO. 16 gacacgtgcatatgagcatg

[35] 1.3 Experimental method

[36] S1, peripheral blood circulating tube cells (peripheral blood CTC) were intercepted with a filter membrane.

[37] 1.5 ml of a peripheral blood sample of a patient was collected by using an EDTA anticoagulant blood collecting vessel, the blood collecting vessel was inverted for uniform mixing, 15 ml of an erythrocyte lysate (SurExam Inc. USA) was added into the blood collecting vessel for uniform mixing, and the blood was kept static at room temperature for 30 min to pyrolyze erythrocytes.

[38] The formula of the erythrocyte lysate included 154 mM NHzCI, 10mM KHCOkU505210 and 0.1mM EDTA.

[39] 2. The mixture was centrifugalized at 500xg for 5 min to remove a blood sample supernatant.

[40] 3. The mixture was resuspended with a PBS (Wuhan Boster Biological

Technology., Co., Ltd., with the article No. ARO030) for cell sedimentation.

[41] 4. Residual cells were fixed with formaldehyde with the final concentration of 4% for sedimentation for 8 min.

[42] 5. The fixed cells were transferred to a filter pipe containing a filter membrane (Becton, Dickinson and Company, 8 pM), and the cells were filtered to the filter membrane with a vacuum suction filtration pump (Tianjin Automatic Science Instrument

Co., Ltd., with the article No. AP-01P).

[43] 6. The filtered filter membrane sampled was continuously fixed at room temperature for 1 h with 4% formaldehyde.

[44] The pore diameter of the filter membrane used in the embodiment was 8 uM, and the peripheral blood CTC was intercepted by a filtration method of the filter membrane (ISET method) to effectively remove leukocytes and intercept the peripheral blood CTC.

[45] S2, typing detection was performed on the intercepted peripheral blood CTC with three specific capture probes.

[46] 1. The fixed filter membrane sample was washed three times with the PBS and was placed in a 24-well plate.

[47] 2. 0.1 mg/ml protease K (Sigma, St. Louis, USA, CAS No.: 39450-01-6) was added for treatment, and the sample was kept static at room temperature for 1 h to increase the membrane permeability.

[48] 3. The sample was washed three times with the PBS to differentiate types of the circulating tumor cells, and the three specific capture probes were added for hybridization.

[49] The three specific capture probes (specific sequences were shown in table 3) were respectively:

[50] (1) the epitheliated peripheral blood CTC specific capture probes EpCAM, CK8,

CK18, and CK19; (2) the interstitial peripheral blood CTC specific capture probé&/505210

[51] A hybridization reaction was performed at 40°C for 3 h. Uncombined specific capture probes were washed three times with 1000 ul of an eluent. The formula of the eluent included: 0.1xsaline sodium citrate (SSC) (Thermo, with the article No. AM9765).

[52] 4. 100 pl of a pre-amplification solution was added.

[53] The formula of the pre-amplification solution included: 30% equinum serum, 1.5% lauryl sodium sulfate (Sigma, St. Louis, USA, with the article No. L5750-500G), 3 mM Tris-HCI (pH 8.0) (Sigma, St. Louis, USA, with the article No. T3038-1L), and 0.5 fmol of a pre-amplification probe (the sequence was shown in table 4), and the pre-amplification solution was put at 40°C for incubation for 30 min to carry out a signal expansion probe reaction.

[54] 5. The membrane was cooled, was eluted three times (0.1xSSC) with 1000 pl of eluent, and was mixed with 100 pl of an amplification solution, and 1 fmol of the pre-amplification probe (the sequence was shown in table 4) for incubation at 40°C for 30 min.

[55] The formula of the amplification solution included: 30% equinum serum, 1.5% lauryl sodium sulfate, and 3 mM Tris-HCI (pH 8.0).

[56] 6. Three marked fluorescent proteins were added, which were respectively fluorescent dies Alexa Fluor 594 (for marking the epitheliated peripheral blood CTC specific capture probes EpCAM and CK8/18/19, Alexa Fluor 488 (for marking the interstitial peripheral blood CTC specific capture probes Vimentin and Twist), and Alexa

Fluor 750 (for marking the leukocyte phenotypic biomarker CD45), and the marked fluorescent proteins were put at 40°C for incubation for 30 min.

[57] 7. The sample was eluted with 0.1xSSC, then nuclear staining was performe«&/505210 with DAPI (SIGMA, with the article No. S26939) for 5 min, and the sample was observed with an automatic fluorescent scanning microscope under a 100-multiple oil immersion lens.

[58] Experimental results are shown in FIG. 1, where red dotted fluorescence represents expression conditions of epithelial markers of peripheral blood CTC: EpCAM,

CK8, CK18 and CK19 genes; green dotted fluorescence represents expression conditions of interstitial markers: Vimentin and Twist genes of peripheral blood CTC; a white signal point represents a gene expression condition of a leukocyte marker CD45; and The interpretative standard herein needs not to be divided into high, medium and low expression levels. If the number of the fluorescent signal points is greater than 7, it is determined as positive.

[59] Table 3 Sequences of nucleic acid probes LU505210

SEQ ID NO. 17 TGGTGCTCGTTGATGAGTCA

SEQ ID NO. 18 AGCCAGCTTTGAGCAAATGA

SEQ ID NO. 19 AAAGCCCATCATTGTTCTGG

FPCAM SEQ ID NO. 20 CTCTCATCGCAGTCAGGATC

SEQ ID NO. 21 TCCTTGTCTGTTCTTCTGAC

SEQ ID NO. 22 CTCAGAGCAGGTTATTTCAG

SEQ ID NO. 23 CGTACCTTGTCTATGAAGGA

SEQ ID NO. 24 ACTTGGTCTCCAGCATCTTG

SEQ ID NO. 25 CCTAAGGTTGTTGATGTAGC cre SEQ ID NO. 26 CTGAGGAAGTTGATCTCGTC

SEQ ID NO. 27 CAGATGTGTCCGAGATCTGG

SEQ ID NO. 28 TGACCTCAGCAATGATGCTG

SEQ ID NO. 29 AGAAAGGACAGGACTCAGGC

SEQ ID NO. 30 GAGTGGTGAAGCTCATGCTG

SEQ ID NO. 31 TCAGGTCCTCGATGATCTTG cre SEQ ID NO. 32 CAATCTGCAGAACGATGCGG

SEQ ID NO. 33 AAGTCATCAGCAGCAAGACG

SEQ ID NO. 34 CTGCAGTCGTGTGATATTGG

SEQ ID NO. 35 CTGTAGGAAGTCATGGCGAG

SEQ ID NO. 36 AAGTCATCTGCAGCCAGACG

SEQ ID NO. 37 CTGTTCCGTCTCAAACTTGG

CK SEQ ID NO. 38 TTCTTCTTCAGGTAGGCCAG

SEQ ID NO. 39 CTCAGCGTACTGATTTCCTC

SEQ ID NO. 40 GTGAACCAGGCTTCAGCATC

SEQ ID NO. 41 GAGCGAGAGTGGCAGAGGAC

Vimentin SEQ ID NO. 42 CTTTGTCGTTGGTTAGCTGG

SEQ ID NO. 43 CATATTGCTGACGTACGTCA

SEQ ID NO. 44 GAGCGCCCCTAAGTTTTTAA

SEQ ID NO. 45 AAGATTGCAGGGTGTTTTCG

SEQ ID NO. 46 GGCCAATAGTGTCTTGGTAG

SEQ ID NO. 47 ACAATGACATCTAGGTCTCC

SEQ ID NO. 48 CTGGTAGAGGAAGTCGATGT

SEQ ID NO. 49 CAACTGTTCAGACTTCTATC

Twist SEQ ID NO. 50 CCTCTTGAGAATGCATGCAT

SEQ ID NO. 51 TTTCAGTGGCTGATTGGCAC

SEQ ID NO. 52 TTACCATGGGTCCTCAATAA

SEQ ID NO. 53 TCGCAATTCTTATGCGACTC

SEQ ID NO. 54 TGTCATGGAGACAGTCATGT

SEQ ID NO. 55 GTATTTCCAGCTTCAACTTC

Chas SEQ ID NO. 56 CCATCAATATAGCTGGCATT

SEQ ID NO. 57 TTGTGCAGCAATGTATTTCC

SEQ ID NO. 58 TACTTGAACCATCAGGCATC

[60] Table 4 Sequences of bDNA signal amplification probes LU505210 bDNA probe Functional Serial Sequence (5’-3’) Remark bDNA probe for Capture SEQ ID NO. CTACAAACAAACAATATT Preamplifier

EpCAM and probe tail 59 CGCAGCCTCAGCC leader (1)

CK8/18/19 (1) SEQ ID NO. CCCAGACCCTACC Amplifier leader

Preamplifier 60 (1) repeat (5) SEQ ID NO. Label probe (1)

Amplifier 61 repeat (5) bDNA probe for Capture SEQ ID NO.CTTCTCAATAACTAACAT Preamplifier

Vimentin and probe tail 62 GACGGTCGGCGTT leader (1)

Twist (1) SEQ ID NO. GTCACCGCTCCAC Amplifier leader

Preamplifier 63 (1) repeat(5) SEQ ID NO. Label probe (1)

Amplifier 64 repeat (5) bNDA probe for Capture SEQ ID NO. GTAAAAAGAAAGGTATAA Preamplifier

CD45 probe tail 65 AATTATACATCTC leader (1) (1) SEQ ID NO. GAAATGAATGAAT Amplifier leader

Preamplifier 66 (1) repeat (5) SEQ ID NO. Label probe (1)

Amplifier 67 repeat (5)

[61] Remark: a bDND probe is associated with a CTC typing detection technical platform.

[62] Kits used in the experiment, manufacturers, and article No. are as follows: LU505210

[63] CD45 detection kit, Surexam Bio-Tech Co., Ltd, with the article No. 22030301.

EP-CAM detection kit, Surexam Bio-Tech Co., Ltd, with the article No. 22030302.

Cytokeratin 8 kit, Surexam Bio-Tech Co., Ltd, with the article No. 22030303. Cytokeratin 18 kit, Surexam Bio-Tech Co., Ltd, with the article No. 22030304. Cytokeratin 19 kit,

Surexam Bio-Tech Co., Ltd, with the article No. 22030305. Vimentin/twist detection kit,

Surexam Bio-Tech Co., Ltd, with the article No. 22030306.

[64] SZ’, the expression condition of MRNA of PHLPP was detected in the peripheral blood CT with a PHLPP probe.

[65] The step is substantially the same as the detection method in S2, which includes the following specific steps:

[66] 1. The fixed filter membrane sample was washed three times with the PBS and was placed in a 24-well plate.

[67] 2. 0.1 mg/ml protease K (Sigma, St. Louis, USA, CAS No.: 39450-01-6) was added for treatment, nd the mixture was kept static at room temperature for 1 h to increase the membrane permeability.

[68] 3. The sample was washed three times with the PBS, and the PHLPP probe was added for a hybridization reaction at 40°C for 3 h. Uncombined PHLPP probes were washed three times with 1000 ul of an eluent. The formula of the eluent included: 0.1xSSC.

[69] 4. 100 pl of a pre-amplification solution was added.

[70] The formula of the pre-amplification solution included: 30% equinum serum, 1.5% lauryl sodium sulfate, 3 mM Tris-HCI (pH 8.0), and 0.5 fmol of a pre-amplification probe (the sequence was shown in table 4), and the pre-amplification solution was put at 40°C for incubation for 30 min to carry out a signal expansion probe reaction.

[71] 5. The membrane was cooled, was eluted three times (0.1xSSC) with 1000 pl of eluent, and was mixed with 100 pl of an amplification solution, and 1 fmol of the pre-amplification probe (the sequence was shown in table 4) for incubation at 40°C for 30 min.

[72] The formula of the amplification solution included: 30% equinum serum, 1.5%

lauryl sodium sulfate, and 3 mM Tris- HCI (pH 8.0). LU505210

[73] 6. A fluorescent dye Alexa Fluor 647 (marked as purple) was added, and the sample was put at 40°C for incubation for 30 min.

[74] 7. The sample was eluted with 0.1xSSC, then nuclear staining was performed with DAPI for 5 min, and the sample was observed with an automatic fluorescent scanning microscope under a 100-multiple oil immersion lens.

[75] The purple signal points represented expression of the PHLPP gene, and the high, medium, and low expression levels of the PHLPP were divided according to the number of the signal points.

[76] Table 5 Division of medium, and low expression levels of PHLPP

Gene Type points 0 No expression 1-3 Low expression

PHLPP

4-9 Medium expression [771 The experimental results are shown in FIG. 1: the detection method in the embodiment can predict the sensitivity of the patient to chemotherapeutic drugs such as

Oxaliplatin, 5-Fluorouracil, and Capecitabine before treatment, and can dynamically detect and evaluate drug resistance of the tumor cells in the treatment process, so as to observe the time points when chemotherapy resistance is generated.

[78] CTC is one of the quite important detection items in liquid biopsy. Regardless of shape, source, and component, CTC is most consistent with tissue biopsy. Therefore,

CTC can be repeatedly acquired by collecting peripheral blood, and detection of MRNA of the PHLPP in CTC can dynamically monitor changes of the chemosensitivity of the patients with colon cancer.

[79] Through the designed PHLPP probe, the mRNA level of PHLPP1 and PHLPP2 #505210 the peripheral blood CTC of the patent with colon cancer is detected by means of filtration by a nano membrane in combination with the mRNA in-situ hybridization technique, and it is found that the chemosensitivity of the patients with high mRNA level of PHLPP1 and PHLPP2 in CTC is good, and the curative effect of chemotherapy for the patients with low mRNA level of PHLPP1 and PHLPP2 in CTC is poor. Therefore, the

PHLPP probe and the detection method therefor provided by the present invention are adopted to detect the mRNA level of PHLPP in the peripheral blood CTC to better predict the chemosensitivity of the patent before treatment.

[80] The primary cause to detect the mRNA level of the PHLPP of peripheral blood

CTC by means of the mRNA in-situ hybridization technique lies in that

[81] (1) CTC and tissue samples are highly consistent, sampling can be repeated, and the time point when drug resistance will occur on the patients can be captured timely; and (2) the mRNA in situ hybridization features high specificity, high sensitivity, easy and rapid operation, and low cost.

[82] Example 2

[83] The example provides a detection kit for identifying mRNA expression levels of

PHLPP in peripheral blood with different phenotypes, specifically including the following components:

[84] Table 6 Components of detection kit LU505210

Component name Main components box

Erythrocyte lysate NH4CL, KHCOs3, EDTA 30% equinum serum, 1.5% lauryl sodium

Pre-amplification sulfate, 3mM Tris-HCI (pH 8.0), 0.5 fmol solution pre-amplification probe (the sequence is shown in table 4)

BOX 1 30% equinum serum, 1.5% lauryl sodium

Amplification solution sulfate, 3mM Tris-HCI (pH 8.0)

Fluorescent dyes Alexa Fluor 594, Alexa

Fluorescent proteins

Fluor 488, Alexa Fluor 750

Specific capture probes SEQ ID NO.17 to SEQ ID NO.58 0.1 x SSC /

EDTA anticoagulant blood collecting vessel /

BOX 2 Filter tube containing a filter membrane with the / pore diameter of 8 uM

[85] The detection method of the kit refers to S1 and S2, which is not described repeatedly herein.

[86] Example 3

[87] The example provides a kit for detecting the sensitivity of a chemotherapeutic drug for CRC, which can predict the sensitivity of the patient to chemotherapeutic drugs such as Oxaliplatin, 5-Fluorouracil, and Capecitabine before treatment, and can dynamically detect and evaluate drug resistance of the tumor cells in the treatment process, so as to observe the time points when chemotherapy resistance is generated.

Specific components of the kit are shown in the following table:

[88] Table 7 Components of detection kit LU505210

Packaging

Component name Main components box

Erythrocyte lysate NH4CL, KHCOs3, EDTA 30% equinum serum, 1.5% lauryl sodium sulfate, 3mM Tris-HCI (pH 8.0), 0.5 fmol

Erythrocyte lysate pre-amplification probe (the sequence is shown in table 4) 30% equinum serum, 1.5% lauryl sodium

BOX 1 Amplification solution sulfate, 3mM Tris-HCI (pH 8.0)

Fluorescent proteins Fluorescent dyes Alexa Fluor 647

PHLPP probe for

SEQ ID NO.1 to SEQ ID NO.8; SEQ ID detecting PHLPP1 and

NO.9 to SEQ ID NO.16

PHLPP2 0.1 x SSC /

EDTA anticoagulant / blood collecting vessel

BOX 2 Filter tube containing a filter membrane with the / pore diameter of 8 uM

[89] The detection method of the kit refers to S1 and S2'.

[90] 5-fluorouracil (5-FU) is a first-line chemotherapeutic drug for tumors such as CRC and gastric cancer, which slows down biological synthesis of DNA by inhibiting the activity of thymine nucleotide synthetase, so as to achieve the purpose of inhibiting tumor cell proliferation. Clinical tests verify that 5-FU has a better curative effect, but the individual responses of the patients differ greatly, even with adverse reactions. The activity of the key enzyme: methylenetetrahydrofolate reductase (MTHFR) of a folate metabolic pathway of the patient is usually detected in the prior art to predict the chemosensitivity of human tumor cells to 5-FU. Capecitabine is an anti-metaboli¢)505210 fluoropyrimidine deoxyribonucleoside carbamate drug capable of being converted in vivo into 5-FU. Oxaliplatin is a first-line chemotherapeutic drug for treating patients with CRC.

However, about 50% of patients with CRC have drug resistance to chemotherapy based on Oxaliplatin.

[91] Example 3

[92] The example provides a detection kit capable of predicting the sensitivity of a patient to chemotherapeutic drugs such as Oxaliplatin, 5-Fluorouracil, and Capecitabine and identifying the expression levels of mRNA of PHLPP in the peripheral blood CTC with different phenotypes. The components of the detection kit as shown in the following table:

[93] Table 8 Components of detection kit LU505210

Packaging

Component name Main components box

Erythrocyte lysate NH4CL, KHCOs3, EDTA 30% equinum serum, 1.5% lauryl sodium sulfate, 3mM Tr is-HCI (pH 8 .0), 0.5 fmol

Erythrocyte lysate pre-amplification probe (the sequence is shown in table 4) 3mM Tr is-HCI(pH 8 .0): 30% equinum

Amplification solution serum, 1.5% lauryl sodium sulfate, 3mM Tr is-HCI (pH 8 .0)

BOX 1 Fluorescent dyes Alexa Fluor 594, Alexa

Fluorescent proteins Fluor 488, Alexa Fluor 750, Alexa Fluor 647

PHLPP probe for

SEQ ID NO.1 to SEQ ID NO.8; SEQ ID detecting PHLPP1 and

NO.9 to SEQ ID NO.16

PHLPP2

Specific capture

SEQ ID NO.17 to SEQ ID NO.58 probes 0.1 x SSC /

EDTA anticoagulant / blood collecting vessel

BOX 2 Filter tube containing a filter membrane with the / pore diameter of 8 uM

[94]

[95] The detection method of the kit refers to S1, S2, and S2’. LU505210

[96] The preferred specific embodiments of the present invention are described in detail above. It should be understood that those of ordinary skill in the art can make various modifications and variations in accordance with concept of the present invention without creative efforts. Therefore, technical solutions capable of being obtained by technicians in the technical field through logical analysis, inference or limited experiments in accordance with concept of the present invention based on the prior art shall fall into the scope of protection determined by claims.

SEQUENCE LISTING LU505210 <110>MARKER PROBE FOR PREDICTING SENSITIVITY OF CHEMOTHERAPEUTIC

DRUG FOR

COLORECTAL CANCER AND APPLICATION

<120>Chongqing University Cancer Hospital <160>67 <170>PatentIn version3.5 <210>1 <211>20 <212>DNA <213>artificial sequence <400>1 gctttttcac ttcttctact 20 <210>2 <211>20 <212>DNA <213>artificial sequence <400>2 gtctgtaagt tgtgcatcac 20 <210>3 <211>20 <212>DNA <213>artificial sequence <400>3 tattgtctcg taggtcaage 20 <210>4 <211>20 <212>DNA <213>artificial sequence

<400>4 LU505210 aaagacctcg atgcagttgg 20 <210>5 <211>20 <212>DNA <213>artificial sequence <400>5 gtgtaaccat gactccatac 20 <210>6 <211>20 <212>DNA <213>artificial sequence <400>6 cgacagagaa ctgtttggca 20 <210>7 <211>20 <212>DNA <213>artificial sequence <400>7 tccgggggta tgataaactg 20 <210>8 <211>20 <212>DNA <213>artificial sequence <400>8 gcctcactat acaacactga 20 <210>9 <211>20 <212>DNA <213>artificial sequence

<400>9 LU505210 cgtttcatat ttctettgge 20 <210>10 <211>20 <212>DNA <213>artificial sequence <400>10 ccaaagtctc gaagctgaca 20 <210>11 <211>20 <212>DNA <213>artificial sequence <400>11 agatttccca attcttcagg 20 <210>12 <211>20 <212>DNA <213>artificial sequence <400>12 tctgagattt aaggccttgg 20 <210>13 <211>20 <212>DNA <213>artificial sequence <400>13 gttccagaac cagatttgta 20 <210>14 <211>20 <212>DNA <213>artificial sequence

<400>14 LU505210 gatagtggct accagtttat 20 <210>15 <211>20 <212>DNA <213>artificial sequence <400>15 tcagagtcat ctgccataac 20 <210>16 <211>20 <212>DNA <213>artificial sequence <400>16 gacacgtgca tatgagcatg 20 <210>17 <211>20 <212>DNA <213>artificial sequence <400>17 tggtgctegt tgatgagtca 20 <210>18 <211>20 <212>DNA <213>artificial sequence <400>18 agccagcttt gagcaaatga 20 <210>19 <211>20 <212>DNA <213>artificial sequence

<400>19 LU505210 aaagcccatc attgttctgg 20 <210>20 <211>20 <212>DNA <213>artificial sequence <400>20 ctctcatcgc agtcaggatc 20 <210>21 <211>20 <212>DNA <213>artificial sequence <400>21 tcettgtetg ttettetgac 20 <210>22 <211>20 <212>DNA <213>artificial sequence <400>22 ctcagagcag gttatttcag 20 <210>23 <211>20 <212>DNA <213>artificial sequence <400>23 cgtaccttgt ctatgaagga 20 <210>24 <211>20 <212>DNA <213>artificial sequence

<400>24 LU505210 acttggtctc cagcatcttg 20 <210>25 <211>20 <212>DNA <213>artificial sequence <400>25 cctaaggttg ttgatgtagce 20 <210>26 <211>20 <212>DNA <213>artificial sequence <400>26 ctgaggaagt tgatctcgtc 20 <210>27 <211>20 <212>DNA <213>artificial sequence <400>27 cagatgtgtc cgagatctgg 20 <210>28 <211>20 <212>DNA <213>artificial sequence <400>28 tgacctcagc aatgatgctg 20 <210>29 <211>20 <212>DNA <213>artificial sequence

<400>29 LU505210 agaaaggaca ggactcaggc 20 <210>30 <211>20 <212>DNA <213>artificial sequence <400>30 gagtggtgaa gctcatgctg 20 <210>31 <211>20 <212>DNA <213>artificial sequence <400>31 tcaggtcctc gatgatcttg 20 <210>32 <211>20 <212>DNA <213>artificial sequence <400>32 caatctgcag aacgatgcgg 20 <210>33 <211>20 <212>DNA <213>artificial sequence <400>33 aagtcatcag cagcaagacg 20 <210>34 <211>20 <212>DNA <213>artificial sequence

<400>34 LU505210 ctgcagtcgt gtgatattgg 20 <210>35 <211>20 <212>DNA <213>artificial sequence <400>35 ctgtaggaag tcatggcgag 20 <210>36 <211>20 <212>DNA <213>artificial sequence <400>36 aagtcatctg cagccagacg 20 <210>37 <211>20 <212>DNA <213>artificial sequence <400>37 ctattecatc tcaaacttgg 20 <210>38 <211>20 <212>DNA <213>artificial sequence <400>38 ttcttcttca ggtaggecag 20 <210>39 <211>20 <212>DNA <213>artificial sequence

<400>39 LU505210 ctcagcgtac tgatttectc 20 <210>40 <211>20 <212>DNA <213>artificial sequence <400>40 gtgaaccagg cttcagcatc 20 <210>41 <211>20 <212>DNA <213>artificial sequence <400>41 gagcgagagt ggcagaggac 20 <210>42 <211>20 <212>DNA <213>artificial sequence <400>42 ctttgtcgtt ggttagetgg 20 <210>43 <211>20 <212>DNA <213>artificial sequence <400>43 catattgctg acgtacgtca 20 <210>44 <211>20 <212>DNA <213>artificial sequence

<400>44 LU505210 gagcgcccct aagtttttaa 20 <210>45 <211>20 <212>DNA <213>artificial sequence <400>45 aagattgcag ggtgttttcg 20 <210>46 <211>20 <212>DNA <213>artificial sequence <400>46 ggccaatagt gtcttggtag 20 <210>47 <211>20 <212>DNA <213>artificial sequence <400>47 acaatgacat ctaggtctcc 20 <210>48 <211>20 <212>DNA <213>artificial sequence <400>48 ctggtagagg aagtcgatgt 20 <210>49 <211>20 <212>DNA <213>artificial sequence

<400>49 LU505210 caactgttca gacttctatc 20 <210>50 <211>20 <212>DNA <213>artificial sequence <400>50 cctcttgaga atgcatgcat 20 <210>51 <211>20 <212>DNA <213>artificial sequence <400>51 tttcagtggc tgattggcac 20 <210>52 <211>20 <212>DNA <213>artificial sequence <400>52 ttaccatggg tcctcaataa 20 <210>53 <211>20 <212>DNA <213>artificial sequence <400>53 tcgcaattct tatgcgactc 20 <210>54 <211>20 <212>DNA <213>artificial sequence

<400>54 LU505210 tgtcatggag acagtcatgt 20 <210>55 <211>20 <212>DNA <213>artificial sequence <400>55 gtatttccag cttcaacttc 20 <210>56 <211>20 <212>DNA <213>artificial sequence <400>56 ccatcaatat agctggcatt 20 <210>57 <211>20 <212>DNA <213>artificial sequence <400>57 ttgtgcagca atgtatttcc 20 <210>58 <211>20 <212>DNA <213>artificial sequence <400>58 tacttgaacc atcaggcatc 20 <210>59 <211>18 <212>DNA <213>artificial sequence

<400>59 LU505210 ctacaaacaa acaatatt 18 <210>60 <211>13 <212>DNA <213>artificial sequence <400>60 cgcagcctca gec 13 <210>61 <211>13 <212>DNA <213>artificial sequence <400>61 cccagaccct acc 13 <210>62 <211>18 <212>DNA <213>artificial sequence <400>62 cttctcaata actaacat 18 <210>63 <211>13 <212>DNA <213>artificial sequence <400>63 gacggtcggce gtt 13 <210>64 <211>13 <212>DNA <213>artificial sequence

<400>64 LU505210 gtcaccgctc cac 13 <210>65 <211>18 <212>DNA <213>artificial sequence <400>65 gtaaaaagaa aggtataa 18 <210>66 <211>13 <212>DNA <213>artificial sequence <400>66 aattatacat ctc 13 <210>67 <211>13 <212>DNA <213>artificial sequence <400>67 gaaatgaatg aat 13

Claims

CLAIMS LU505210

1. A marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC, comprising a PHLPP probe for detecting PHLPP1 and PHLPP2, wherein the nucleotide sequence of the probe for detecting PHLPP1 is one or a combination of SEQ ID NO. 1 to SEQ ID NO. 8, and the nucleotide sequence of the probe for detecting PHLPP2 is one or a combination of SEQ ID NO. 9 to SEQ ID NO. 16.

2. An application of the probe according to claim 1 in preparation of a detection kit.

3. A method for detecting the marker probe for predicting the sensitivity of a chemotherapeutic drug for CRC according to claim 1 or 2, comprising the following steps: S1, intercepting peripheral blood CTC with a filter membrane; and S2’, detecting the expression condition of mRNA of PHLPP in the peripheral blood CT with a PHLPP probe.

4. An application of the detection method according to claim 3 in preparation of a detection kit.

5. A kit for detecting the sensitivity of a chemotherapeutic drug for CRC, comprising a PHLPP probe for detecting PHLPP1 and PHLPP2.