Circular RNA molecule for detecting rectal cancer radiotherapy sensitivity and application of circular RNA molecule

Abstract

The invention relates to a circular RNA molecule for detecting rectal cancer radiotherapy sensitivity and application of the circular RNA molecule, and discloses a molecular marker circDNAJC3 for detecting colorectal cancer radiotherapy sensitivity. The expression of the circDNAJC3 in a biological sample of a patient sensitive to colorectal cancer radiotherapy is up-regulated, the patient sensitive to radiotherapy can be conveniently and accurately predicted in advance by detecting the expression level of the circDNAJC3 in colorectal cancer tissue of the patient, the treatment strategy can beoptimized, medical resources are reasonably utilized, and accurate treatment is achieved. The application of the circDNAJC3 is based on clinical research, the molecular mechanism of the circDNAJC3 isresearched and confirmed, and the clinical application value is extremely high.

Description

technical field

[0001] The invention relates to the technical field of clinical medicine, in particular to a circular RNA molecule for detecting radiotherapy sensitivity of rectal cancer and an application thereof. Background technique

[0002] In 2018, the number of new cases and deaths of colorectal cancer in my country ranked fifth in the world, and 70% of non-metastatic patients were in the middle and locally advanced stages. According to current international guidelines, these patients need preoperative radiotherapy. After preoperative radiotherapy, most patients with rectal cancer can be downstaged, but most of them cannot achieve clinical complete remission (cCR), and still need to complete surgery, and preoperative radiotherapy will reduce the overall complications of surgery to a certain extent. If patients who are sensitive to radiotherapy can be predicted in advance, it can help to optimize treatment strategies, rationally utilize medical resources, and implement ...

Examples

Embodiment 1

[0046] Example 1 Screening of differentially expressed circRNAs

[0047] Modeling 12 rectal cancer patients, 5 of them achieved pathological complete remission after preoperative radiotherapy (radiotherapy sensitive group, code G), and 7 cases were postoperative pathologically graded 3-4 (Mandard grading standard, radiotherapy resistant group, code U). Clinical samples of colorectal cancer tissues from each patient were collected, and total RNA was extracted from all clinical sample tissues by the trizol method and qualified for quality control before library construction and sequencing. Specific steps are as follows:

[0048] (1) Extract total RNA:

[0049] Take the tissue out of the liquid nitrogen, transfer it to a 1.5mL RNase-free EP tube, add 1ml Trizol lysate immediately, use a tissue homogenizer to grind and lyse the tissue cells, and wait until the liquid is cloudy and there is no obvious tissue block at 12,000rpm Centrifuge for 5 minutes, and transfer the supernata...

Embodiment 2

[0054] Example 2 qRT-PCR sequencing verification

[0055] Based on the 119 circRNAs with significantly different expression levels obtained in Example 1, circDNAJC3 among them was selected for verification in this example. Specific steps are as follows:

[0056] (1) Extract tissue RNA

[0057] According to the method of Example 1, the clinical tissues of 28 patients (including 14 cases in the radiotherapy-sensitive group and 14 cases in the radiotherapy-resistant group) who had undergone preoperative radiotherapy were collected, and RNA was extracted respectively.

[0058] (2) cDNA synthesis

[0059] 1. RNA pre-denaturation:

[0060] Add the following reaction liquids to the microcentrifuge tube, as shown in Table 1:

[0061] Table 1

[0062] Reagent Dosage RNA 2.0μg Random / Oligo primer 1μL wxya 2 o

Make up to 16μL

[0063] Incubate at 65°C for 5 minutes, and immediately place the mixture on ice for 2 minutes;

[0064] 2. RNA rev...

Embodiment 3

[0084] Example 3 Transcriptome sequencing verification

[0085] This embodiment is further extended to 35 independent specimens (including 6 cases in the radiotherapy-sensitive group and 29 cases in the radiotherapy-resistant group) for transcriptome sequencing, and the specific steps are as follows:

[0086] One: miRNA sequencing experiment process

[0087] (1) Nucleic acid extraction: collect and extract RNA according to the method of Example 1

[0088] (2) Library construction

[0089] 1. Reagents: Multiplex Small RNA Library Prep Kit

[0090] 2. Instrument: 2720Thermal Cycler (life Technologies), Agilent Technologies 2200 TapeStation (life Technologies)

[0091] 3. miRNA library construction steps:

[0092] 3.1 3' end connector connection:

[0093] Take total RNA 100ng-1μg, total volume 6μL. Add 1 μL 3’SR Adapter for Illumina by pipetting and mixing, and place in a PCR instrument for 2 minutes at 70°C. After the reaction was completed, 10 μL 3’Ligation ReactionBuf...

technical field

[0001] The invention relates to the technical field of clinical medicine, in particular to a circular RNA molecule for detecting radiotherapy sensitivity of rectal cancer and an application thereof. Background technique

[0002] In 2018, the number of new cases and deaths of colorectal cancer in my country ranked fifth in the world, and 70% of non-metastatic patients were in the middle and locally advanced stages. According to current international guidelines, these patients need preoperative radiotherapy. After preoperative radiotherapy, most patients with rectal cancer can be downstaged, but most of them cannot achieve clinical complete remission (cCR), and still need to complete surgery, and preoperative radiotherapy will reduce the overall complications of surgery to a certain extent. If patients who are sensitive to radiotherapy can be predicted in advance, it can help to optimize treatment strategies, rationally utilize medical resources, and implement ...

Examples

Embodiment 1

[0046] Example 1 Screening of differentially expressed circRNAs

[0047] Modeling 12 rectal cancer patients, 5 of them achieved pathological complete remission after preoperative radiotherapy (radiotherapy sensitive group, code G), and 7 cases were postoperative pathologically graded 3-4 (Mandard grading standard, radiotherapy resistant group, code U). Clinical samples of colorectal cancer tissues from each patient were collected, and total RNA was extracted from all clinical sample tissues by the trizol method and qualified for quality control before library construction and sequencing. Specific steps are as follows:

[0048] (1) Extract total RNA:

[0049] Take the tissue out of the liquid nitrogen, transfer it to a 1.5mL RNase-free EP tube, add 1ml Trizol lysate immediately, use a tissue homogenizer to grind and lyse the tissue cells, and wait until the liquid is cloudy and there is no obvious tissue block at 12,000rpm Centrifuge for 5 minutes, and transfer the supernata...

Embodiment 2

[0054] Example 2 qRT-PCR sequencing verification

[0055] Based on the 119 circRNAs with significantly different expression levels obtained in Example 1, circDNAJC3 among them was selected for verification in this example. Specific steps are as follows:

[0056] (1) Extract tissue RNA

[0057] According to the method of Example 1, the clinical tissues of 28 patients (including 14 cases in the radiotherapy-sensitive group and 14 cases in the radiotherapy-resistant group) who had undergone preoperative radiotherapy were collected, and RNA was extracted respectively.

[0058] (2) cDNA synthesis

[0059] 1. RNA pre-denaturation:

[0060] Add the following reaction liquids to the microcentrifuge tube, as shown in Table 1:

[0061] Table 1

[0062] Reagent Dosage RNA 2.0μg Random / Oligo primer 1μL wxya 2 o

Make up to 16μL

[0063] Incubate at 65°C for 5 minutes, and immediately place the mixture on ice for 2 minutes;

[0064] 2. RNA rev...

Embodiment 3

[0084] Example 3 Transcriptome sequencing verification

[0085] This embodiment is further extended to 35 independent specimens (including 6 cases in the radiotherapy-sensitive group and 29 cases in the radiotherapy-resistant group) for transcriptome sequencing, and the specific steps are as follows:

[0086] One: miRNA sequencing experiment process

[0087] (1) Nucleic acid extraction: collect and extract RNA according to the method of Example 1

[0088] (2) Library construction

[0089] 1. Reagents: Multiplex Small RNA Library Prep Kit

[0090] 2. Instrument: 2720Thermal Cycler (life Technologies), Agilent Technologies 2200 TapeStation (life Technologies)

[0091] 3. miRNA library construction steps:

[0092] 3.1 3' end connector connection:

[0093] Take total RNA 100ng-1μg, total volume 6μL. Add 1 μL 3’SR Adapter for Illumina by pipetting and mixing, and place in a PCR instrument for 2 minutes at 70°C. After the reaction was completed, 10 μL 3’Ligation ReactionBuf...

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