Application of long-chain non-coding RNA in breast cancer

Abstract

The invention provides application of long-chain non-coding RNA in breast cancer, and belongs to the field of biological detection. The tissue-derived long-chain non-coding RNA LINC02660-201 is closely related to the breast cancer and can be used as an RNA molecular marker for clinical diagnosis. According to ROC curve analysis, the AUC is 0.9259, the sensitivity is 88.89%, and the specificity is 84.80%. The expression level of the gene in a breast cancer sample is obviously reduced, and the gene is related to poorer clinical pathological parameters, and is related to HER2 expression, lower ER and PR expression levels, higher Ki-67 expression level, higher histological classification and more invasive molecular typing.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to an application of long-chain non-coding RNA LINC02660-201 in breast cancer. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Breast cancer is a malignant tumor that seriously threatens women's health. It has a high incidence and mortality rate worldwide. Early detection and diagnosis of breast cancer is extremely important. Breast ultrasound is one of the commonly used methods, but it may be affected by the operator's technical level and experience, and may not be accurate enough in identifying small lesions. Mammography has a relatively poor diagnostic effect on dense breasts. Although magnetic resonance imaging (MRI) has a high sensitivity in the diagnosis of breast cancer, it is expensive, takes a long time to examine, and may overdiagnose certain benign lesions. Therefore, finding minimally invasive tumor markers with both high sensitivity and high specificity to guide clinical practice and achieve early detection and diagnosis of breast cancer has always been an important topic in breast cancer research.

[0004] Long non-coding RNA (LncRNA) is a class of non-coding RNA molecules with a length of more than 200 nucleotides. They can interact with DNA, RNA or protein, affect the structure and function of chromatin, and thus regulate gene transcription. They can also regulate the stability, splicing and translation of mRNA at the post-transcriptional level. A large amount of evidence shows that the abnormal expression of lncRNA in cancer cells is accompanied by DNA damage, immune escape and cellular metabolic disorders, leading to the progression of various cancers. lncRNA will show specific expression patterns under different physiological and pathological conditions, and the stability of lncRNA is relatively high. Moreover, lncRNA can participate in the regulation of various cellular processes and signaling pathways. Therefore, specific types of lncRNA have the potential to become biomarkers. Summary of the invention

[0005] The present invention screened and obtained tissue-derived lncRNA, which is closely related to breast cancer and can be used as an RNA molecular marker for clinical diagnosis. The RNA molecular marker can distinguish breast cancer tissue and its adjacent tissue with high sensitivity and specificity, and has excellent diagnostic performance.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] The present invention provides an application of a reagent for detecting the expression level of a long-chain non-coding RNA in the preparation of a product for screening, diagnosing, detecting, monitoring breast cancer or predicting the progression of breast cancer, wherein the long-chain non-coding RNA is Lnc432452.

[0008] Optionally, the long non-coding RNA is derived from breast tissue or breast cancer tissue.

[0009] Optionally, the reagent includes a primer for the long non-coding RNA.

[0010] Optionally, the sequence of the primer is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0011] Optionally, the reagent further includes an internal reference gene, and the internal reference gene is the GAPDH gene.

[0012] Optionally, the reagent also includes primers for internal reference genes.

[0013] Optionally, the primer of the internal reference gene is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0014] Optionally, the product includes RNA extraction reagents, reverse transcription reagents and / or PCR amplification reagents.

[0015] Optionally, the application includes histological grading and / or molecular typing of breast cancer.

[0016] Optionally, the molecular typing includes Luminal A type, Luminal B type, Her-2 positive type and triple negative type.

[0017] One or more of the above technical solutions have the following beneficial effects:

[0018] The present invention demonstrates for the first time the feasibility and clinical utility of LINC02660-201 as a biomarker for breast cancer and for predicting its malignancy. ROC curve analysis showed that LINC02660-201 can well distinguish breast cancer tissue and its adjacent tissue, with an area under the curve (AUC) of 0.9259, a sensitivity of 88.89%, and a specificity of 84.80%. The expression level of LINC02660-201 in breast cancer samples was significantly reduced and was associated with worse clinical pathological parameters, which was associated with HER2 expression, lower ER and PR expression levels, higher Ki-67 expression levels, higher histological grades, and more aggressive molecular typing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 Figure 1 is a graph showing the detection results of LncRNA markers in the embodiments of the present invention; A: a graph showing the results of whole transcriptome sequencing screening and identification, in which upregulated or downregulated lncRNAs were obtained; B: a graph showing the results of PCR analysis of Lnc511443, which was significantly downregulated in breast cancer compared with adjacent tissues; C: a graph showing the results of PCR analysis of Lnc525328, which was significantly downregulated in breast cancer compared with adjacent tissues; D: a graph showing the results of PCR analysis of Lnc569740, which was expressed below the set detection limit in most tissues and had no statistically significant difference in expression between the two groups; E: a graph showing the results of PCR analysis of Lnc432452, which was significantly downregulated in breast cancer compared with adjacent tissues; F: a graph showing the results of PCR analysis of Lnc511443, which was significantly downregulated in breast cancer compared with adjacent tissues; 443 PCR analysis results, the expression in 50 breast cancer tissues was significantly lower than that in adjacent tissues; G: Lnc525328 PCR analysis results, the expression in 50 breast cancer tissues was significantly lower than that in adjacent tissues; H: Lnc432452 PCR analysis results, the expression in 50 breast cancer tissues was significantly lower than that in adjacent tissues; I: Lnc432452 PCR analysis results, the expression in 171 breast cancer tissues was significantly lower than that in adjacent tissues; J: Real-time quantitative PCR analysis results of lnc432452 (AUC = 0.9259, P < 0.001), with a sensitivity of 88.89% and a specificity of 84.80%.

[0021] Figure 2The following are statistical results of the diagnosis and detection of breast cancer using Lnc432452 in the embodiment of the present invention; A: Histological grading result diagram, the expression level of Lnc432452 gradually decreases from grade I, grade II to grade III; B: Estrogen receptor (ER) detection result diagram, the expression of Lnc432452 in PR-positive patients is higher than that in negative patients; C: Progesterone receptor (PR) detection result diagram, the expression of Lnc432452 in PR-positive patients is higher than that in negative patients; D: Human epidermal growth factor receptor-2 (HER2) detection result diagram, the expression level of Lnc432452 in HER2-positive patients is lower than that in HER2-negative patients; E: Ki-67 expression level detection result diagram, the expression level of Lnc432452 is lower in patients with higher Ki-67 expression levels; F: Luminal subtype and non-luminal subtype detection result diagram, the Luminal subtype has a higher level of Lnc432452 expression than the non-luminal subtype; G: Luminal A, Luminal B, the test results of Her-2 positive and triple negative; Luminal A vs Luminal B, P = 0.0242; Luminal A vs Her-2 positive, P < 0.0001; Luminal A vs triple negative, P = 0.0037; and compared with the Her-2 positive subtype, the expression of Lnc432452 in the Luminal B subtype was higher; H: the test results of luminal subtype, Her-2 positive and triple negative subtype, luminal vs Her-2 positive, P = 0.0003; Luminal vs triple negative, P = 0.0237. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0023] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0024] In the present invention, the term "treatment" has its general meaning, and in this article specifically refers to the treatment of mammalian individuals (preferably humans) who have been infected with the new coronavirus with the drug of the present invention, in order to produce a therapeutic, curative, alleviating, or mitigating effect on the disease, and obtain the desired pharmacological and / or physiological effects.

[0025] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0026] The gene name of the long noncoding RNA of the breast cancer prognostic marker is LINC02660-201; gene ID: ENST00000432452; hereinafter referred to as Lnc432452.

[0027] Example

[0028] 1. Screening of lncRNA markers related to breast cancer based on high-throughput sequencing

[0029] (1) Clinical samples

[0030] From May 2017 to July 2021, breast cancer patients were recruited at Qilu Hospital of Shandong University, and all patients were Han females.

[0031] Cancer and paracancerous tissues from 3 breast cancer patients were collected for whole transcriptome sequencing screening, and 4 potential differentially expressed lncRNAs were obtained for subsequent verification; cancer and paracancerous tissues from 30 breast cancer patients were used for preliminary qRT-PCR verification; cancer and paracancerous tissues from 50 breast cancer patients were used for further qRT-PCR verification; and cancer and paracancerous tissues from 171 breast cancer patients were used for large-sample qRT-PCR verification. All breast cancer and paracancerous tissues were obtained during surgery. All participants signed written informed consent. This study was approved by the Ethics Committee of Qilu Hospital of Shandong University.

[0032] (2) RNA extraction

[0033] 1) Take out 30-50 mg of tissue specimens stored in liquid nitrogen and quickly transfer them to an EP tube. Add 700 μL of RNA extraction reagent (RNA-easy Isolation Reagent) and use tissue scissors to fully chop the tissue until it becomes a paste without obvious visible particles to form a lysis solution. Add 280 ml of RNase-free double distilled water (RNase-free ddH2O) to the above lysis solution, mix it upside down, let it stand at room temperature for 5 minutes, and then centrifuge it at 12,000 rpm for 15 minutes at room temperature.

[0034] 2) Take out the centrifuge tube. The solution is now separated into layers, including an upper aqueous phase containing RNA and a lower dark precipitate containing impurities such as protein, DNA, and polysaccharides. Carefully pipette the upper aqueous phase into a new centrifuge tube.

[0035] 3) Add an equal volume of isopropanol to the centrifuge tube containing the upper aqueous phase, mix by inverting, and let stand for 10 minutes.

[0036] 4) Centrifuge the centrifuge tube containing the upper aqueous phase and isopropanol at 12,000 rpm for 10 min at room temperature to obtain a white precipitate, and carefully discard the supernatant.

[0037] 5) Add 1000 μL of 75% v / v ethanol (prepared with RNase-free ddH2O) to the EP tube containing the white precipitate, gently tap the bottom of the tube to suspend the precipitate, and invert it several times.

[0038] 6) Centrifuge the tube containing ethanol at 8,000 rpm for 3 min at room temperature, discard the supernatant, and air dry at room temperature. Note: Do not over-dry, otherwise the RNA sample will be difficult to dissolve.

[0039] 7) Add a set amount of RNase-free ddH2O to the dried EP tube to dissolve the precipitate, and vortex at room temperature for 3 minutes to fully dissolve the RNA precipitate.

[0040] 8) Measure the concentration and purity of RNA: first add RNase-free ddH2O to zero, then take 1 μL of the dissolved RNA solution, detect the RNA concentration on a micro-spectrophotometer and record it; an OD260 / OD280 ratio between 1.8-2.2 indicates high purity; RNA is stored in a -80℃ refrigerator when not in use.

[0041] (3) Reverse transcription of RNA

[0042] 1) Genomic DNA removal

[0043] Prepare a mixed solution with the composition shown in Table 1 in an RNase-free centrifuge tube.

[0044] Table 1

[0045] Reagents Usage <![CDATA[RNase-free ddH2O]]> No more than 16 μL 4-fold dilution of gDNA wiper Mix 4μL Template RNA 1pg-1μg

[0046] The template RNA is the RNA obtained in the RNA extraction in step (2). The mixture is gently blown and mixed with a pipette, and reacted at 42° C. for 2 min to obtain 16 μL of the first reaction solution.

[0047] 2) Prepare the reverse transcription reaction system

[0048] Add 5-fold diluted HiScript II qRTSuperMix II reagent directly to the reaction tube containing the first reaction solution to a total volume of 20 μL, and mix by gently pipetting.

[0049] 3) Perform reverse transcription reaction

[0050] The reverse transcription reaction was carried out at 50°C for 15 minutes, followed by an enzyme inactivation reaction at 85°C for 5 seconds. After the reaction, the reaction product was stored at -20°C.

[0051] (4) Real-time quantitative polymerase chain reaction (qRT-PCR)

[0052] 1) Thaw and mix the solutions required for various PCR reactions and place them on ice.

[0053] 2) Prepare the PCR reaction system according to the components and volumes in Table 2, then gently tap the bottom of the tube to mix the solution, and briefly centrifuge at 5000 rpm to obtain a mixed solution.

[0054] Table 2

[0055]

[0056]

[0057] 3) Add 5.4ul of the mixed solution to each well of the PCR plate, add the set amount of cDNA, and use ddH2O to make up the liquid in each well to 10μL. Place the prepared PCR plate on ice before setting the PCR program.

[0058] 4) Place the PCR plate in step 3) on a Realtime PCR instrument for PCR reaction. All indicators are performed according to the following program: Stage 1: 95°C, 30 s; Stage 2: 40 PCR cycles (95°C, 3-10 s; 60°C, 10-30 s); Stage 3: 95°C, 15 s; 60°C, 60 s; 95°C, 15 s.

[0059] 5) Melting curve analysis, using GAPDH as the internal reference gene to standardize lncRNA expression, the primers used are shown in Table 3, including the upstream primer of Lnc432452 as shown in SEQ ID NO.1, the downstream primer of Lnc432452 as shown in SEQ ID NO.2, the downstream primer of GAPDH gene as shown in SEQ ID NO.3 and the downstream primer of GAPDH gene as shown in SEQ ID NO.4, and then the results were used 2 -△Ct Method calculation.

[0060] Table 3

[0061]

[0062] (5) Whole transcriptome sequencing

[0063] Ribo-Zero rRNA Removal Kits (Illumina, USA) were used to remove rRNAs from total RNA. TruSeq Stranded Total RNA Library Prep Kit (Illumina, USA) was used to pretreat RNA and construct sequencing libraries. The BioAnalyzer 2100 instrument (Agilent Technologies, USA) was used for library quality control and quantification.

[0064] According to the Illumina sequencing instructions, 10pM library was denatured into single-stranded DNA molecules, captured on Illumina flowcell, amplified in situ into clusters, and sequenced for 150 cycles using the double-end mode (PE mode) on the Illumina HiSeq sequencer. After sequencing on the Illumina HiSeq4000 sequencer, double-end reads were harvested. Q30 was used for quality control, and cutadapt software (v1.9.3) was used to remove adapters and low-quality reads to obtain high-quality reads. High-quality reads were aligned to the human reference genome (UCSC HG19) using hisat2 software (v2.0.4). Then, under the guidance of the gtf gene annotation file, the FPKM (Fragments per kilobase of exon per million fragments mapped) value of LncRNA at the transcript level was obtained using cuffdiff software (v2.2.1, part of the cufflinks software suite) as the expression profile of LncRNA, and the fold change and p-value between two / group samples were calculated to screen differentially expressed LncRNAs.

[0065] (6) Statistical analysis

[0066] Statistical analysis was performed using GraphPad Prism 8 (San Diego, CA). Student t test was used to analyze the differences between the two groups. Chi-square test was used to analyze the relationship between lncRNA expression levels and clinical case parameters. Receiver operating characteristic (ROC) curve analysis was used to clarify the diagnostic value of lncRNA. P < 0.05 was considered statistically significant.

[0067] Based on the whole transcriptome sequencing screening of 3 pairs of breast cancer and adjacent tissue samples in (1) clinical samples, two upregulated lncRNAs were identified, namely ENST00000525328 (named as lnc525328) and ENST00000511443 (named as lnc511443), and two downregulated lncRNAs were identified, namely ENST00000569740 (named as lnc569740) and ENST00000432452 (named as lnc432452) ( Figure 1 (A, P < 0.05, fold difference > 2).

[0068] In the 30 pairs of qRT-PCR preliminary validation tissue samples in (1) clinical samples, real-time quantitative PCR analysis was performed. The expression of ENST00000569740 was extremely low, and the expression in most tissues (22 / 30) was below the set detection limit. Its expression was only detected in 8 pairs of breast cancer and adjacent tissues, but there was no statistical difference in expression between the two groups ( Figure 1 D), so ENST00000569740 was screened out;

[0069] The expression of ENST00000511443, ENST00000525328 and ENST00000432452 in most tissues was higher than the set detection limit, and there was a statistically significant difference in expression between the two groups (respectively Figure 1 As shown in B, C and E, P < 0.0001), ENST00000511443, ENST00000525328 and ENST00000432452 were selected for further verification.

[0070] In (1) 50 pairs of qRT-PCR in clinical samples, qRT-PCR analysis was further performed on tissue samples. The expression of ENST00000511443, ENST00000525328 and ENST00000432452 in 50 breast cancer tissues was significantly lower than that in adjacent tissues (respectively). Figure 1 As shown in F, G, and H, P < 0.0001). However, the expression trends of ENST00000511443 and ENST00000525328 were inconsistent with the whole transcriptome sequencing results (the whole transcriptome sequencing results showed upregulation, but the qRT-PCR results showed downregulation), so ENST00000432452 was selected for subsequent experiments.

[0071] Real-time quantitative PCR analysis was performed on 171 pairs of qRT-PCR large sample validation tissue samples in (1) clinical samples. The results showed that Lnc432452 was significantly downregulated in breast cancer tissues compared with adjacent adjacent tissues ( Figure 1I in; P<0.0001), which has a good diagnostic value in distinguishing breast cancer tissue from adjacent tissue ( Figure 1 The sensitivity of the 3D-type 2-antibody was 88.89% and the specificity was 84.80%.

[0072] 2. Diagnosis and detection of breast cancer using Lnc432452

[0073] The analysis results of the relationship between Lnc432452 expression level and clinical pathological characteristics of breast cancer patients showed that Lnc432452 expression was closely correlated with a variety of clinical pathological parameters.

[0074] (1) Histological grading

[0075] In histological grading, breast cancer is divided into grades I, II, and III. The higher the grade, the worse the biological behavior of breast cancer and the worse the prognosis. The expression level of Lnc432452 gradually decreases from grade I, II, to III ( Figure 2 Among them, the expression of Lnc432452 in patients with grade I histological grade was higher than that in patients with grade III histological grade ( Figure 2 A in the figure, grade I vs grade III, P = 0.0109); the results of the chi-square test showed that the expression of Lnc432452 was also negatively correlated with histological grading (Table 4, P = 0.049), indicating that Lnc432452 can be used for histological grading.

[0076] (2) Molecular typing

[0077] The expression of Lnc432452 in estrogen receptor (ER)-positive patients was higher than that in estrogen receptor-negative patients ( Figure 2 B, P = 0.0055); the expression of Lnc432452 in progesterone receptor (PR) positive patients was higher than that in negative patients ( Figure 2 C in , P = 0.0002); the results of chi-square test showed that the expression of Lnc432452 was negatively correlated with both estrogen receptor (ER) and progesterone receptor (PR) (Table 4, P = 0.007; P = 0.018).

[0078] In breast cancer tissues of HER2-positive patients, the expression level of Lnc432452 was lower than that in breast cancer tissues of HER2-negative patients ( Figure 2 D in (P=0.0015). Meanwhile, the results of chi-square test showed that the expression level of Lnc432452 was negatively correlated with the expression of HER2 (Table 4, P=0.001).

[0079] Patients with higher Ki-67 expression levels had lower Lnc432452 expression levels ( Figure 2The chi-square test also showed that the expression level of Lnc432452 was negatively correlated with the expression level of Ki-67 (Table 4, P<0.001).

[0080] Therefore, Lnc432452 also showed differential expression in different molecular subtypes of breast cancer. Among them, the luminal subtype had a higher level of Lnc432452 expression than the non-luminal subtype ( Figure 2 F, P<0.0001). Compared with the other three subtypes, the expression of Lnc432452 in the luminal A subtype was significantly upregulated ( Figure 2 G, Luminal A vs Luminal B, P = 0.0242; Luminal A vs Her-2 positive, P < 0.0001; Luminal A vs triple negative, P = 0.0037). Compared with the Her-2 positive subtype, Lnc432452 was more highly expressed in the Luminal B subtype ( Figure 2 The expression of Lnc432452 in the luminal subtype was also higher than that in the Her-2 positive and triple negative subtypes ( Figure 2 H in the figure, luminal vs Her-2 positive, P = 0.0003; luminal vs triple negative, P = 0.0237).

[0081] The above shows that Lnc432452 can be used for molecular typing.

[0082] However, the expression level of Lnc432452 was not associated with other clinicopathological characteristics of breast cancer patients, including age, tumor diameter, lymph node metastasis, and TNM stage (Table 4).

[0083] Table 4. Correlation between Lnc432452 expression and clinicopathological characteristics of breast cancer patients

[0084]

[0085]

[0086] *P<0.05.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the expression level of long non-coding RNA in the preparation of a product for screening, diagnosing, detecting, monitoring breast cancer or predicting the progression of breast cancer, characterized in that: The long non-coding RNA is Lnc432452.

2. The use according to claim 1, characterized in that The long non-coding RNA is derived from breast tissue or breast cancer tissue.

3. The use according to claim 1, characterized in that The reagents include primers for the long non-coding RNA.

4. The use according to claim 3, characterized in that The sequence of the primer is shown as SEQ ID NO.1 or SEQ ID NO.

2.

5. The use according to claim 1, characterized in that The reagent also includes an internal reference gene, and the internal reference gene is a GAPDH gene.

6. The use according to claim 5, characterized in that The reagents also include primers for internal reference genes.

7. The use according to claim 6, characterized in that The primer of the internal reference gene is shown in SEQ ID NO.3 or SEQ ID NO.

4.

8. The use according to claim 1, characterized in that The product includes RNA extraction reagents, reverse transcription reagents and / or PCR amplification reagents.

9. The use according to claim 1, characterized in that The applications include histological grading and / or molecular typing of breast cancer.

10. The use according to claim 9, characterized in that The molecular typing includes Luminal A type, Luminal B type, Her-2 positive type and triple negative type.

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