Expression and application of midkine mRNA (messenger ribose nucleic acid) in peripheral blood of patient suffering from lung cancer

A midkine, peripheral blood technology, applied in DNA/RNA fragments, recombinant DNA technology, microbial determination/examination, etc., can solve the problems of low long-term survival rate and poor prognosis, and achieve the effect of excellent diagnostic value

Inactive Publication Date: 2015-04-29
HUZHOU CENT HOSPITAL
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for about 80% of them. Although the curative effec...

Method used

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  • Expression and application of midkine mRNA (messenger ribose nucleic acid) in peripheral blood of patient suffering from lung cancer
  • Expression and application of midkine mRNA (messenger ribose nucleic acid) in peripheral blood of patient suffering from lung cancer
  • Expression and application of midkine mRNA (messenger ribose nucleic acid) in peripheral blood of patient suffering from lung cancer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Design primers, probes, and establish a Taqman probe Real-Time PCR method for detecting MKmRNA:

[0046] (1) Culture lung cancer cell A549, extract total RNA, synthesize cDNA,

[0047] (2) Design PCR amplification primers to amplify and obtain the amplified fragments of the target gene MK and the internal reference gene GAPDH;

[0048] MK primers:

[0049] MK-For: 5'-CGCGGATCCATGCAGCACCGAGGCTTC-3'

[0050] MK-Rev: 5'-CCCAAGCTTCTAGTCCTTTCCCTTCCCTTTC-3'

[0051] GAPDH primer sequence:

[0052] GAPDH-For: 5'-CCGGAATTCCTTCGCTCTCTGCTCCTCCTG-3'

[0053] GAPDH-Rev: 5'-CCGCTCGAGCTCTTCCTCTTGTGCTCTTGCTG-3'

[0054] (3) Transformation, linking, screening, and sequencing of PCR fragments to construct plasmids pET28a-MK and pET28a-GAPDH;

[0055] (4) Design Real-Time PCR primer probes

[0056] MK primers and probes:

[0057] MK-For: 5'-CATCACACGCACCCCAGTT-3'

[0058] MK-Probe: 5'-CTTGCAGTCGGCTCCAAACTCCTTCTT-3'

[0059] MK-Rev: 5'ATTGCGGCGTGGGTTTC-3'

[0060] GAPDH primers...

Embodiment 2

[0070] The expression level of MK mRNA in the peripheral blood of lung cancer patients, LEL patients and normal people was detected by Taqman probe Real-Time PCR method:

[0071] Specimen into the group

[0072] A total of 87 NSCLC patients admitted to Huzhou Central Hospital from June 2009 to December 2011 were selected, including 55 males and 32 females, aged (65.6±15.8) years; 48 cases of squamous cell carcinoma, 35 cases of adenocarcinoma, There were 4 cases of large cell carcinoma; 23 cases of TNM stage Ⅰ-Ⅱ, 64 cases of Ⅲ-Ⅳ stage. All cases were diagnosed as NSCLC with complete clinical data, and none of them received chemotherapy or radiotherapy. Taking 35 LEL patients and 30 healthy volunteers who were admitted to our hospital during the same period as controls, there was no significant difference in general data between the groups (P>0.05). The experimental study was approved by the Ethics Committee of our hospital, and all patients signed informed consent.

[0073]...

Embodiment 3

[0085] (1) The expression level of MK mRNA in the peripheral blood of NSCLC patients, LEL patients, and normal people;

[0086] The expression level of MK mRNA in the peripheral blood of NSCLC patients was 7.20E-03 (IQR: 3.82E-03-12.25E-03), compared with LEL patients (3.69E-03 (IQR: 2.89E-03-5.77E-03)), Compared with normal people (3.05E-03 (IQR: 2.68E-03-4.01E-03)), there are significant differences (both (P0.05). See the attached figure 1 .

[0087] (2) The correlation between the expression level of MK mRNA in peripheral blood and the clinicopathological features of NSCLC

[0088]See Table 1, the expression of MK mRNA is not significantly correlated with gender, age, smoking, tumor size, and tumor histological type of NSCLC patients, but is significantly correlated with TNM stage, degree of differentiation, and lymph node metastasis. The expression level of MK mRNA in stage Ⅲ-Ⅳ NSCLC 8.52 (4.00-17.13) E-03 was significantly higher than that in stage Ⅰ-Ⅱ (4.99 (3.51-7.7...

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Abstract

The invention discloses a fluorescence quantitative PCR (polymerase chain reaction) detection method for MK (midkine) mRNA (messenger ribose nucleic acid) in peripheral blood. According to the method, expression levels of MK mRNA in peripheral blood of a patient suffering from NSCLC (non-small cell lung cancer), a patient suffering from LEL (lung benign lesion) and a healthy examined person are compared, correlation between the expression levels and NSCLC clinical and pathological characteristics is analyzed, feasibility of taking expression of MK mRNA in peripheral blood as an NSCLC differential and diagnostic marker is discussed, a result indicates that the expression levels of MK mRNA in peripheral blood of the patient suffering from NSCLC, the patient suffering from LEL and the healthy examined person are remarkably different, and high expression levels are remarkably associated with clinical staging, lymphatic metastasis and cell differentiation degree of NSCLC. Therefore, detection of MK mRNA in peripheral blood can be applied to effective detection of differential diagnosis of NSCLC.

Description

technical field [0001] The invention relates to the application of midkine mRNA in cancer diagnosis, especially the application of midkine mRNA in peripheral blood for differential diagnosis of non-small cell lung cancer. Background technique [0002] Lung cancer is one of the most common malignant tumors in my country, and its morbidity and mortality rank first among malignant tumors. Non-small cell lung cancer (Non-Small Cell Lung Cancer, NSCLC) accounts for about 80% of them. Although the curative effect has improved in recent years, the long-term survival rate is still relatively low. With palliative treatment, the prognosis is poor. [0003] Midkine (MK) is a secreted heparin-binding growth factor, which is a low molecular weight protein. MK is abundantly expressed in the second trimester and plays an important role in the development of pregnancy. In adults, MK is restricted to express in certain tissues. However, in the process of tumorigenesis, inflammation and t...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/158C12Q2600/166
Inventor 马志红李丽琴黄惠莲李静戴利成
Owner HUZHOU CENT HOSPITAL
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