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Screening method for serum exosome marker of laryngeal squamous carcinoma and application for exosome source miR-941

A technology of mir-941 and laryngeal squamous cell carcinoma, which is applied in the field of molecular diagnosis and molecular biology, can solve the problems of patients suffering and inability to diagnose early, and achieve reliable diagnostic results, great clinical practical value, and simple and fast clinical diagnosis

Pending Publication Date: 2020-06-05
FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to solve the defects in the prior art that the diagnosis of laryngeal squamous cell carcinoma still requires histological examination, which causes pain to patients and cannot be diagnosed early, the present invention provides a method for screening serum exosomal miRNA markers of laryngeal squamous cell carcinoma

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  • Screening method for serum exosome marker of laryngeal squamous carcinoma and application for exosome source miR-941
  • Screening method for serum exosome marker of laryngeal squamous carcinoma and application for exosome source miR-941
  • Screening method for serum exosome marker of laryngeal squamous carcinoma and application for exosome source miR-941

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Experimental program
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Embodiment 1

[0032] Serum exosome extraction, identification and exosome RNA extraction:

[0033] Step 1, collection of serum samples

[0034] Collection objects: A total of 87 serum samples were collected, including 56 histopathologically confirmed laryngeal squamous cell carcinoma patients and 31 healthy controls. Patients with squamous cell carcinoma of the larynx were from the Department of Otolaryngology, Head and Neck Surgery, the First Hospital of Shanxi Medical University, and had no history of radiotherapy and chemotherapy or acute and chronic inflammatory diseases, and were used as the experimental group. The healthy control subjects were from the physical examination center of the hospital, had no history of acute and chronic inflammatory diseases and malignant tumors, and matched the age and sex of the experimental group. This study was approved by the Research Ethics Committee of Shanxi Medical University, and all subjects signed informed consent.

[0035] Specific steps: co...

Embodiment 2

[0045] Screening of differential miRNAs in serum exosomes of patients with laryngeal squamous cell carcinoma and healthy controls by RNA sequencing:

[0046] Serum exosomal RNA from 6 patients with laryngeal squamous cell carcinoma and 6 healthy controls was used for RNA sequencing analysis in this experiment.

[0047] Step 1, sample detection, library construction and sequencing

[0048] (1) Total RNA sample detection: The high-sensitivity Agilent 2100pic600 is used to accurately detect the total amount and fragment distribution of RNA.

[0049] (2) Library construction: After the sample is qualified, use the Small RNA Sample Pre Kit to construct the library, using the special structure of the 3' and 5' ends of Small RNA (the 5' end has a complete phosphate group, and the 3' end has a hydroxyl group), Using total RNA as the starting sample, directly add adapters to both ends of Small RNA, and then reverse transcribe to synthesize cDNA. Subsequently, after PCR amplification,...

Embodiment 3

[0055] Real-time fluorescence quantitative (qRT-PCR) verification of serum exosome differences in patients with laryngeal squamous cell carcinoma and healthy controls:

[0056] There is no recognized stable internal reference gene in serum exosomes. In this experiment, the internal reference gene was first screened and identified, and then the qRT-PCR verification of the differentially expressed miRNAs in Experiment 2 was carried out. In this experiment, serum exosomal RNA of 50 patients with laryngeal squamous cell carcinoma and 25 healthy controls was used for qRT-PCR verification of differentially expressed miRNAs, among which, serum exosomal RNA from 7 patients with laryngeal squamous cell carcinoma and 7 healthy controls Body RNA was also used for qRT-PCR detection in internal reference screening. The specific implementation steps are as follows:

[0057] Step 1, internal reference gene screening

[0058] The present invention screens the serum exosomal RNAs stably expr...

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Abstract

The invention belongs to the technical fields of molecular diagnosis and molecular biology, and specifically relates to a screening method for a serum exosome marker of laryngeal squamous carcinoma and an application for an exosome source miR-941. The screening method comprises the following steps: screening serum exosome miRNAs which are differentially expressed between a laryngeal squamous carcinoma patient and a healthy control person through high-throughput sequencing, and selecting miRNAs which are up-regulated and expressed in the laryngeal squamous carcinoma patient and are high in expression quantity as candidate miRNAs; carrying out qRT-PCR verification on the candidate miRNAs in another group of expanded samples, and screening out a reference gene for qRT-PCR detection before verification; and analyzing the diagnosis efficiency of target miRNAs by adopting an ROC curve. The invention also provides a serum exosome miRNA marker miR-941 for the laryngeal squamous carcinoma. Thus, a new way is provided for minimally invasive diagnosis of the laryngeal squamous carcinoma; meanwhile, the serum exosome miRNA marker miR-941 can serve as a potential drug treatment target and has great clinical practical value.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis and molecular biology, and specifically relates to a screening method for serum exosome markers of laryngeal squamous cell carcinoma and the application of miR-941 derived from exosomes. Background technique [0002] Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant tumors of the head and neck, with a high incidence in northern China, including Shanxi Province. In the past 40 years, the incidence of laryngeal squamous cell carcinoma has gradually decreased, while the 5-year survival rate has not increased but decreased, from 66% to 63%. The early symptoms of laryngeal squamous cell carcinoma are not obvious, and about 60% of patients are not diagnosed until the advanced stage (III or IV stage). Early diagnosis can increase the chance of successful treatment, but the anatomical location of the larynx is hidden, so early detection of laryngeal cancer is very d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/686C12N15/113
CPCC12Q1/6886C12Q1/686C12Q2600/158C12Q2561/113C12Q2563/107C12Q2545/114C12Q2521/107
Inventor 高伟赵沁丽吴勇延王斌全徐伟郑希望薛绪亭张春明郭慧娜刘红亮
Owner FIRST HOSPITAL OF SHANXI MEDICAL UNIV
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