Molecular markers of liver cancer and liver cancer detection kit and application

Abstract

The present invention discloses a molecular marker for liver cancer and a liver cancer detection kit and application. In the present invention, RNA sequencing is performed on liver cancer patient tissues in advance to screen lncRNAs with differential expression related to liver cancer, and two lncRNAs are newly found to be abnormally expressed in liver cancer patient tissues. A detection method for the two lncRNAs is established and verified in the blood of liver cancer patients, indicating that the two lncRNAs can be used for liver cancer diagnosis, treatment effect evaluation or prognosis evaluation.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor molecular markers, and in particular relates to molecular markers of liver cancer and a kit for detecting liver cancer and its application. Background Art

[0002] Primary liver cancer is one of the common multiple malignant tumors, ranking third in the world after gastric cancer and esophageal cancer. Primary liver cancer is divided into hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and mixed liver cancer, among which HCC is the most common, accounting for about 85%-90%. Liver cancer is a difficult-to-treat malignant tumor with the strongest heterogeneity and the highest mortality rate. The mortality rate of liver cancer in my country ranks second among tumors, second only to liver cancer. It is currently believed that the occurrence and development of HCC is a complex process regulated by multiple factors, multiple genes, and multiple steps, which is mostly caused by the interaction of genetic and environmental factors. Therefore, in-depth understanding of the molecular mechanisms of tumor occurrence and development, and supplementing and improving the existing theoretical system of cell carcinogenesis, will help provide an important theoretical basis for the prevention, diagnosis and treatment of liver cancer.

[0003] In recent years, due to the rapid development of the second-generation sequencing technology RNA-seq, people have a deeper understanding of non-coding RNA, including long non-coding RNA (lncRNA). LncRNA is a hot topic of recent research. It has many characteristics, such as long length > 200nt, large number, rich secondary structure, and specific but non-protein coding transcripts. With the improvement of research level, scholars have found that lncRNA has strong regulatory functions.

[0004] The sensitivity of the currently known liver cancer molecular markers is low, which is very unfavorable for the early diagnosis of liver cancer. It is of great significance to develop an early diagnosis method for liver cancer. Summary of the invention

[0005] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0006] As one aspect of the present invention, the present invention provides a molecular marker for liver cancer, wherein: the nucleic acid sequence of the molecular marker is one or both of SEQ ID NO: 1 and SEQ ID NO: 2, and the molecular marker is a long non-coding RNA.

[0007] As another aspect of the present invention, the present invention provides a liver cancer detection kit, wherein the liver cancer detection kit comprises primers for detecting the nucleic acid sequence shown in SEQ ID NO: 1 and / or SEQ ID NO: 2.

[0008] As a preferred embodiment of the liver cancer detection kit of the present invention: the primer sequences for detecting the nucleic acid sequence shown in SEQ ID NO: 1 are: upstream primer: 5'-TGGTATTTGCAAGGGACCCA-3', downstream primer: 5'-TGGCTTTTCCCCACTCTAGT-3'.

[0009] As a preferred embodiment of the liver cancer detection kit of the present invention: the primer sequences for detecting the nucleic acid sequence shown in SEQ ID NO: 1 are: upstream primer: 5'-ACCCTGAACACACACCTTCA-3', downstream primer: 5'-TCTCCCTGTGGTCTTTTCCC-3'.

[0010] As another aspect of the present invention, the present invention provides use of the molecular marker of liver cancer in preparing a liver cancer detection kit.

[0011] Preferably, the detection sample of the liver cancer detection kit is a human blood sample.

[0012] Beneficial effects of the present invention: In the present invention, RNA sequencing is performed on liver cancer patient tissues in advance to screen differentially expressed lncRNAs related to liver cancer, and two new lncRNAs are found to be abnormally expressed in liver cancer patient tissues. A detection method for the two lncRNAs is established and verified in the blood of liver cancer patients, indicating that the two lncRNAs can be used for liver cancer diagnosis, treatment effect evaluation or prognosis evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0014] Figure 1 The expression of novel lncRNA-1 and novel lncRNA-2 in the blood of liver cancer patients and normal people.

[0015] Figure 2 This is the ROC curve of novel lncRNA-1 and novel lncRNA-2 for the diagnosis of liver cancer. DETAILED DESCRIPTION

[0016] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0017] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0018] The present invention preliminarily performs RNA sequencing on liver tissues of liver cancer patients, screens differentially expressed lncRNAs related to liver cancer, and verifies them in the serum of normal people and liver cancer patients, and newly discovers two lncRNAs that show abnormal expression in the serum of liver cancer patients. The two newly discovered lncRNAs are named novel lncRNA-1 and novel lncRNA-2, respectively.

[0019] The primer sequences of the two novel lncRNA-1 and novel lncRNA-2 are:

[0020]

[0021] novel lncRNA-1 sequence (SEQ ID NO:1):

[0022]

[0023] Novel lncRNA-2 sequence (SEQ ID NO:2):

[0024]

[0025] Step 1 RNA extraction:

[0026] The blood to be tested comes from normal people and liver cancer patients, and the Trizol method is used to extract blood RNA from normal people and liver cancer patients.

[0027] Step 2: RNA reverse transcription

[0028] The reverse transcription reaction system is as follows:

[0029]

[0030] Step 3: qPCR operation

[0031] The qPCR reaction system is as follows:

[0032]

[0033]

[0034] The reverse transcription reaction conditions were: 37°C for 15 min, 85°C for 5 s, and 4°C infinity; the fluorescence quantitative PCR reaction conditions were: stage 1: 95°C for 30 s, 1 cycle; stage 2: 95°C for 10 s, 60°C for 30 s, 40 cycles; stage 3: 95°C for 15 s, 60°C for 30 s, 95°C for 15 s, and 1 cycle.

[0035] Experimental results:

[0036] Data processing: The present invention uses the ABI 7300 PCR instrument to amplify the target gene with the optimal PCR program and perform data export processing to obtain the melting curves of the two new genes. GraphPad was used to draw the gene expression graph. All statistical analyses were performed in the R language and statistical computing environment (R version 2.15.1; R Foundation for Statistical Computing).

[0037] The results of fluorescence quantitative PCR showed that compared with the normal group, the expression levels of novel lncRNA-1 and novel lncRNA-2 in the blood of liver cancer patients were significantly increased (p < 0.001, p < 0.001). Figure 1 . Figure 1 A and B in the figure are the expression of novel lncRNA-1 and novel lncRNA-2 in the blood of normal people and liver cancer patients, respectively.

[0038] The ROC curves drawn by IVDstatistics showed that the AUC areas of novel lncRNA-1 and novel lncRNA-2 were 0.86 and 0.89, respectively. The sensitivity and specificity of novel lncRNA-1 were 86.67% and 73.33%, respectively. The sensitivity and specificity of novel lncRNA-2 were 93.33% and 76.67%, respectively. Figure 2 . Figure 2 A and B are ROC curves of novel lncRNA-1 and novel lncRNA-2, respectively. It can be seen that the two novel lncRNA-1 and novel lncRNA-2 screened by the present invention have high sensitivity, which is significantly improved compared with the lncRNA in the prior art. Among them, the sensitivity of novel lncRNA-2 reaches 93.33%, which solves the problem of low sensitivity of liver cancer molecular markers in the prior art.

[0039] The present invention has the following advantages: the expression of novel lncRNA-1 and novel lncRNA-2 in the blood of liver cancer patients and normal people is determined, and further verified. The detection of novel lncRNA-1 and novel lncRNA-2 is achieved through the optimal system of fluorescent quantitative PCR, and the results show that novel lncRNA-1 and novel lncRNA-2 have certain diagnostic significance and can be used for liver cancer diagnosis, treatment effect evaluation or prognosis evaluation.

[0040] The present invention identifies lncRNA associated with the progression of liver cancer and explores its function and mechanism. We collected blood samples from 30 liver cancer patients and 30 normal subjects. The selection criteria for liver cancer patients are as follows: (1) patients diagnosed by pathological examination (all patient materials are obtained by microscopy, biopsy or surgery, and confirmed by microscopic examination); (2) patients have no other history of tumors. The tissue was collected during the patient's diagnosis and before any treatment (surgery or chemotherapy). The experimental method of the present invention is to extract RNA, reverse transcribe, and perform fluorescence quantitative PCR on the blood of 30 liver cancer patients and 30 normal subjects, and then verify it. Analysis of the data results showed that compared with the normal group, the expression levels of novel lncRNA-1 and novel lncRNA-2 in the blood of liver cancer patients were significantly upregulated (p < 0.001, p < 0.001), see Figure 1 The above results illustrate the expression of novel lncRNA-1 and novel lncRNA-2 in the blood of patients with liver cancer, and can be used as indicators for the diagnosis and prognosis of liver cancer.

[0041] The present invention has the following advantages: the expression of novel lncRNA-1 and novel lncRNA-2 in the blood of liver cancer patients and normal people is determined, and further verified. The detection of novel lncRNA-1 and novel lncRNA-2 is achieved by the optimal system of fluorescent quantitative PCR, and the results show that novel lncRNA-1 and novel lncRNA-2 have diagnostic significance and can be used for liver cancer diagnosis, treatment effect evaluation or prognosis evaluation. The novel lncRNA-1 and novel lncRNA-2 screened by the present invention have high diagnostic sensitivity for liver cancer, and are of great significance for the early screening and diagnosis of liver cancer.

[0042] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A molecular marker for diagnosing liver cancer, characterized in that: The molecular marker has a nucleic acid sequence of SEQ ID NO: 2, and the molecular marker is a long non-coding RNA.

2. A liver cancer detection kit, characterized in that: The liver cancer detection kit comprises primers for detecting the nucleic acid sequence shown in SEQ ID NO:

2.

3. The liver cancer detection kit according to claim 2, characterized in that: The primer sequences for detecting the nucleic acid sequence shown in SEQ ID NO: 2 are: upstream primer: 5'-ACCCTGAACACACACCTTCA-3', downstream primer: 5'-TCTCCCTGTGGTCTTTTCCC-3'.

4. Use of the molecular marker for diagnosing liver cancer according to claim 1 in preparing a liver cancer detection kit.

Images