Joint detection method for serum markers miR-135a and CEA for detecting early non-small cell lung cancer

Abstract

The invention provides a method for jointly detecting serum markers miR-135a and CEA for detecting early non-small cell lung cancer, the detection method is more sensitive and higher in specificity, and is beneficial to early diagnosis of non-small cell lung cancer and improvement of prognosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a combined detection method for detecting serum markers of early non-small cell lung cancer, which has higher accuracy and sensitivity and is helpful for the early diagnosis of patients with early non-small cell lung cancer. Background Art

[0002] Lung cancer is one of the most common and aggressive malignancies in the world, and is also one of the leading causes of cancer-related deaths. According to the World Health Organization's classification, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer (NSCLC), of which non-small cell lung cancer accounts for about 85% of total lung cancer cases. Currently, complete resection surgery is the most effective treatment for early non-small cell lung cancer. However, it is reported that due to the lack of specific symptoms in the early stage, most patients with non-small cell lung cancer have entered the advanced stage at the time of diagnosis, resulting in poor prognosis. Early screening and treatment are essential to improve the prognosis of patients with non-small cell lung cancer. Serum carcinoembryonic antigen (CEA) is widely accepted in the early diagnosis of non-small cell lung cancer. The diagnostic specificity of serum CEA for non-small cell lung cancer may exceed 90%, but its sensitivity is relatively low, resulting in missed diagnosis and delayed treatment. Therefore, non-invasive and sensitive biomarkers are needed for early detection of non-small cell lung cancer.

[0003] MicroRNA (miRNA) is a class of endogenous, conserved, short-chain non-coding RNAs with a length of about 22 nucleotides. To date, thousands of miRNAs have been identified as tumor suppressors or oncogenes. They can regulate most human genes at the post-transcriptional level by binding to the 30 untranslated regions of target genes. An increasing number of studies have shown that miRNAs play an important role in a variety of biological activities such as proliferation, invasion, metastasis, differentiation, apoptosis and survival of cells or organisms. The expression patterns of miRNAs show specificity for cell types, tissues and developmental stages. Dysregulated miRNAs can affect multiple signaling pathways through their target genes, leading to human diseases such as cancer.

[0004] miRNAs are stable and present in large quantities in body fluids such as blood, urine, and saliva, so they can be detected by simple and non-invasive methods. Therefore, miRNAs are considered promising biomarkers in cancer diagnosis. MiR-135a is a common member of the miRNA family, and studies have shown its dysregulation in NSCLC. Therefore, the expression of miR-135a is significantly downregulated in NSCLC tissues, and its restored expression can reduce the migration and invasion ability of lung cancer cells. MiR-135a may play a tumor suppressor role in non-small cell lung cancer.

[0005] Related studies have shown that compared with healthy individuals, the level of serum miR-135a in NSCLC patients is significantly lower and closely related to disease progression. Serum miR-135a may be a potential biomarker for NSCLC, and the level of serum miR-135a may be related to the inhibition, development and progression of NSCLC. Therefore, the combined detection of serum miR-135a and serum CEA levels may be helpful for the early diagnosis of NSCLC. Summary of the invention

[0006] The invention provides a serum marker for detecting non-small cell lung cancer and a detection method thereof.

[0007] The technical solution of the present invention:

[0008] 1. Patient sample acquisition

[0009] Twenty samples of patients with non-small cell lung cancer (who did not receive any radiotherapy or chemotherapy) were obtained from ××× Hospital, and another 20 blood donors from the same hospital were collected as healthy controls.

[0010] 2. Real-time quantitative polymerase chain reaction (qRT-PCR)

[0011] Total RNA was extracted from previously prepared serum samples using a miRNA isolation kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The first strand of complementary DNA was synthesized by reverse transcription from total RNA using the Taqman microRNA reverse transcription kit (Applied Biosystems). U6 was used as an internal reference.

[0012] 3. Enzyme-linked immunosorbent assay (ELISA)

[0013] Serum CEA levels were detected by ELISA. The third-generation ELISA kit (CanAg, Canada) was used for detection. The operation was carried out according to the manufacturer's instructions, and each sample was tested three times.

[0014] 4. Statistical analysis

[0015] All data were statistically analyzed using SPSS18.0 software. DETAILED DESCRIPTION

[0016] The technical solution of the present invention will be further described below, but the present invention is not limited thereto.

[0017] Example 1

[0018] 1. Patient sample acquisition

[0019] Twenty samples of patients with early non-small cell lung cancer were obtained from ××× Hospital, and 20 other blood donors from the same hospital were collected as healthy controls. 5 mL of peripheral blood was drawn from each subject, and serum samples were centrifuged and stored at 80°C for subsequent RNA isolation.

[0020] 2. Real-time quantitative polymerase chain reaction (qRT-PCR)

[0021] Total RNA was extracted from previously prepared serum samples using a miRNA isolation kit (Applied Biosystems, Foster City, CA). The first strand of complementary DNA was reverse transcribed from total RNA using the Taqman microRNA reverse transcription kit (Applied Biosystems). U6 was used as an internal reference.

[0022] The primers for miR-135a and U6 are as follows: MIR-135a (forward: 50-ACACTCCAGCTGGGGTATGGCTTTTTATTCCT30; reverse: 50-GGTGTCGTGGAGTCGGCAA-30); U6 (forward: 50-CTCGCTTCGGCAGCACA-30; reverse: 50-AACGCTTCACGAATTTGCGT-30). Gene amplification was performed under the following temperature conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s.

[0023] The PCR amplification conditions for miR-135a were: 95°C for 20 s, 95°C for 10 s, 60°C for 20 s, 40 cycles, and finally incubation for 5 s at 70°C. The expression level of miR-135a was normalized to U6 and determined using the 2DDCt method.

[0024] 3. Enzyme-linked immunosorbent assay (ELISA)

[0025] Serum CEA levels were detected by ELISA. The third-generation ELISA kit (CanAg, Canada) was used for detection. The operation was carried out according to the manufacturer's instructions, and each sample was tested three times.

[0026] 4. Statistical analysis

[0027] All data were statistically analyzed using SPSS 18.0 software. The expression of serum miR-135a and CEA was expressed as mean ± standard deviation, and the difference in the expression of miR-35a in non-small cell lung cancer and healthy control group was analyzed by t test.

[0028] ROC analysis was used to evaluate the diagnostic value of serum miR-135a in NSCLC, and then to explore the diagnostic value of combined detection of miR-35a and CEA in non-small cell lung cancer. Relative ROC curves were drawn to express the relative true positive rate and relative false positive rate, which changed with the positive threshold of biomarkers. P < 0.05 was considered statistically significant.

Claims

1. A method for detecting the serum markers miR-135a and CEA for early non-small cell lung cancer, characterized in that: The following steps are involved: Step 1: Obtain samples from 20 patients with early non-small cell lung cancer, and 20 other blood donors from the same hospital were collected as healthy controls. 5 mL of peripheral blood was drawn from each subject, and serum samples were centrifuged and stored at 80°C for subsequent RNA isolation. Step 2: Real-time quantitative polymerase chain reaction (qRT-PCR) and ELISA were used to detect serum CEA levels, and SPSS18.0 software was used for statistical analysis of the data.

2. The method according to claim 1, characterized in that The lung cancer patients only include patients with stage I and stage II non-small cell lung cancer.

3. The method according to claim 1, characterized in that The experimental specimens in step 1 include, but are not limited to, ADC and SCC patient samples.

4. The detection method according to claim 2, characterized in that: The following steps are involved: Step 1: Real-time quantitative polymerase chain reaction (qRT-PCR) Total RNA was extracted from previously prepared serum samples using a miRNA isolation kit (Applied Biosystems, Foster City, CA). The first strand of complementary DNA was reverse transcribed from total RNA using the Taqman microRNA reverse transcription kit (Applied Biosystems). U6 was used as an internal reference. The primers for miR-135a and U6 are as follows: MIR-135a (forward: 50-ACACTCCAGCTGGGGTATGGCTTTTTATTCCT30; reverse: 50-GGTGTCGTGGAGTCGGCAA-30); U6 (forward: 50-CTCGCTTCGGCAGCACA-30; reverse: 50-AACGCTTCACGAATTTGCGT-30). Gene amplification was performed under the following temperature conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. The PCR amplification conditions for miR-135a were as follows: 95°C for 20 s, 95°C for 10 s, and 60°C for 20 s for 40 cycles, and finally incubated at 70°C for 5 s. The expression level of miR-135a was normalized to U6 and determined using the 2DDCt method. Step 2: Enzyme-linked immunosorbent assay (ELISA) Serum CEA levels were detected by ELISA. The third-generation ELISA kit (CanAg, Canada) was used for detection. The operation was carried out according to the manufacturer's instructions, and each sample was tested three times. Step 3: Statistical analysis All data were statistically analyzed using SPSS 18.0 software. The expression of serum miR-135a and CEA was expressed as mean ± standard deviation, and the difference in the expression of miR-35a in non-small cell lung cancer and healthy control group was analyzed by t test. ROC analysis was used to evaluate the diagnostic value of serum miR-135a in NSCLC, and then to explore the diagnostic value of combined detection of miR-35a and CEA in non-small cell lung cancer. Relative ROC curves were drawn to express the relative true positive rate and relative false positive rate, which changed with the positive threshold of biomarkers. P < 0.05 was considered statistically significant.

5. The detection method according to claim 2 is characterized in that the detection method is more sensitive and has higher specificity, which is helpful for early diagnosis of early non-small cell lung cancer and improving prognosis.