A non-small cell lung cancer-related lncRNA molecular marker and its application

Abstract

The present invention relates to the field of molecular biology technology, and in particular to a lncRNA molecular marker related to non-small cell lung cancer and its application. The lncRNA molecular marker is lncRNA MIF-AS1. Experiments have shown that the expression level of lncRNA MIF-AS1 in the peripheral blood of NSCLC patients and NSCLC cell lines is significantly increased, indicating that lncRNA MIF-AS1 has diagnostic value for NSCLC; at the same time, the predictive value of lncRNA MIF-AS1 for the sensitivity of NSCLC to radiotherapy and chemotherapy is better than the traditional biomarker CEA, indicating that lncRNA MIF-AS1 can be used as a biomarker for the sensitivity of NSCLC to radiotherapy and chemotherapy. The lncRNA MIF-AS1 provided by the present invention provides a new biomarker for the early diagnosis of NSCLC and provides a new direction for further understanding the underlying mechanism of NSCLC.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a lncRNA molecular marker associated with non-small cell lung cancer and applications thereof. Technical Background

[0002] Non-small-cell lung cancer (NSCLC) is the main type of lung cancer, accounting for approximately 80% of all lung cancers. Despite significant advances in treatment options, the prognosis of NSCLC remains poor, with a 5-year survival rate of approximately 15%. One of the reasons for this poor prognosis is that NSCLC lacks obvious clinical manifestations in the early stages, and most patients are diagnosed only when lymph node metastasis and distant metastasis develop. Therefore, exploring new biomarkers that can achieve early diagnosis of NSCLC is an urgent clinical issue that needs to be addressed.

[0003] Long non-coding RNAs (lncRNAs) are evolutionarily conserved non-coding RNAs that play a key role in a variety of human diseases, including cancer. Numerous studies have shown that abnormalities in lncRNAs play a key role in the development, invasion, metastasis, and chemotherapy resistance of non-small cell lung cancer (NSCLC). The expression of lncRNA MNX1-AS1 is significantly upregulated in NSCLC, and abnormal expression of lncRNA MNX1-AS1 is significantly associated with TNM stage, lymph node metastasis, and prognosis of NSCLC. Furthermore, knockdown of lncRNA MNX1-AS1 inhibits the proliferation, migration, and invasion of A549 cells and promotes apoptosis (Liu G, Guo X, Zhang Y, et al. Expression and significance of LncRNA MNX1-AS1 in non-small cell lung cancer [J]. OncoTargets and therapy, 2019, 12:3129).

[0004] Numerous studies have shown significant differences in lncRNA expression profiles between NSCLC and normal lung tissue. Furthermore, due to the stability, ease of detection, cost-effectiveness, and non-invasiveness of lncRNAs, and with the advancement of RNA molecule delivery technology, these lncRNAs may serve as biomarkers for the early diagnosis of NSCLC and provide potential drug targets for its treatment. However, the significance of only a few lncRNAs in NSCLC has been determined. Therefore, further exploration and identification of additional lncRNAs that play a role in NSCLC is of great significance. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a lncRNA molecular marker related to non-small cell lung cancer, the lncRNA molecular marker is lncRNA MIF-AS1, and the sequence is SEQ ID NO.1.

[0006] Another object of the present invention is to provide the use of the lncRNA molecular marker in the preparation of a detection product for screening and diagnosis of non-small cell lung cancer.

[0007] Another object of the present invention is to provide a kit for diagnosing non-small cell lung cancer, which includes primers for detecting the lncRNA molecular markers as described above, or antibodies that specifically bind to the lncRNA molecular markers as described above.

[0008] The present invention also provides a use of the lncRNA molecular marker described above in preparing a reagent for predicting the sensitivity of non-small cell lung cancer to radiotherapy and chemotherapy.

[0009] The beneficial effects of the present invention are:

[0010] This study used lncRNA sequencing to analyze lncRNA expression profiles in peripheral blood samples from NSCLC patients and discovered that a novel lncRNA, MIF-AS1, was significantly expressed in the peripheral blood of NSCLC patients and in NSCLC cell lines. Experiments confirmed that this lncRNA MIF-AS1 has diagnostic value for NSCLC. Furthermore, the predictive value of lncRNA MIF-AS1 for NSCLC chemoradiotherapy sensitivity was superior to that of the traditional biomarker CEA, indicating that lncRNA MIF-AS1 can serve as a biomarker for NSCLC chemoradiotherapy sensitivity.

[0011] The lncRNA MIF-AS1 provided by the present invention provides a new biomarker for the early diagnosis of NSCLC and provides a new direction for further understanding the potential mechanism of NSCLC. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the screening result of differentially expressed lncRNA between sensitive group and resistant group.

[0013] Figure 2 To screen for differential expression of lncRNAs associated with cisplatin sensitivity in NSCLC.

[0014] Figure 3 The RT-qPCR test method was used to verify the expression difference of lncRNA MIF-AS1 between the chemoradiotherapy-sensitive group and the resistant group of NSCLC. **P<0.01.

[0015] Figure 4 This is the ROC curve analysis of the lncRNA MIF-AS1 content in the serum of NSCLC patients and its sensitivity to NSCLC chemoradiotherapy. DETAILED DESCRIPTION

[0016] For ease of understanding, the technical solution of the present invention is described in more detail below with reference to experiments:

[0017] 1. Patients and sample collection

[0018] A total of 61 patients with NSCLC who were admitted to the Thoracic Oncology Center of Hefei Cancer Hospital, Chinese Academy of Sciences, between 2021 and 2022 were included. Patients had NSCLC confirmed by pathological examination of available biopsy specimens (according to the 8th edition of the Union for International Cancer Control (UICC) TNM staging system for lung cancer) and had received prior treatment with radiotherapy, chemotherapy, or other treatments. This study adhered to ethical standards for human research and was approved by the Ethics Committee of Hefei Cancer Hospital, Chinese Academy of Sciences. All subjects voluntarily signed written informed consent before plasma samples were collected.

[0019] 2 experiments

[0020] 2.1 lncRNA assay

[0021] According to the NEBNext Ultra RNA LibraryPrep Kit for Illumina (NEB, USA) kit instructions, RNA fragments were synthesized for first-strand and second-strand cDNA, and then enriched by adapter ligation and low-cycle PCR amplification. The library was quality-checked and quantified using an Agilent 2200 TapeStation and Qubit. The final library that passed the quality inspection was sequenced using an Illumina Nova sequencer (Illumina, USA) at Ribobio Co. Ltd (Ribobio, China) using a double-end 150-bp read length, i.e., PE150. Finally, the data were normalized and analyzed using the edgeR package software. The criteria for differential lncRNAs were an absolute logFC value greater than 0.5 and an FDR less than 0.01.

[0022] 2.2 RT-qPCR assay

[0023] Total RNA was extracted using a viral RNA extraction reagent (TIANGEN, Beijing, China). Total RNA was reverse transcribed into cDNA using an mRNA / lncRNA qRT-PCR Startert Kit (Ruibo, Guangzhou, China) and analyzed using an ABI7300PLUS Fast PCR Real-Time System (Applied Biosystems, Foster City, CA, USA). qPCR was performed using Prepremix ExTaq™II (Takara, Dalian, China). Reaction conditions included pre-denaturation at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 34 seconds. λpolyA+RNA-A was used as an internal control, and data were analyzed using the 2-ΔΔCT method. Primers were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.

[0024] 2.3 Statistical analysis

[0025] Statistical processing, analysis, and plotting were performed using SPSS 21.0 (IBM Corporation, Armonk, NY, USA) and Origin pro 2019 (Origin Lab Corporation, Northampton, MA, USA). The nonparametric Mann-Whitney U test was used to compare the relative expression levels of lncRNA MIF-AS1 in whole blood among patients with NSCLC in different groups. Receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were used to evaluate the diagnostic efficacy of lncRNA MIF-AS1 and CEA for NSCLC. The Youden's index was used to determine the optimal cutoff point.

[0026] 3. Results

[0027] 3.1 lncRNA MIF-AS1 expression is significantly elevated in the blood of NSCLC patients and may serve as a biomarker for NSCLC

[0028] The lncRNA experiment collected 6 clinical plasma samples from patients, of which 3 were sensitive to cisplatin treatment and 3 were resistant. Exosomal DNA was extracted from the plasma, and lncRNA gene sequencing was performed using the second-generation sequencing method. Differential analysis was performed using the edgeR package. With the logFC absolute value greater than 0.5 and the FDR less than 0.01 as the threshold, a total of 5189 differentially expressed lncRNAs were obtained, of which 1629 were downregulated and 3560 were upregulated. Figure 1 shown.

[0029] At the same time, 62 lung adenocarcinoma cell lines and 15 lung squamous cell carcinoma cell lines were downloaded from the GDSC database, of which 17 were cisplatin-sensitive cell lines and 60 were cisplatin-resistant. Differential analysis was performed on the sensitive and resistant groups, and lncRNAs related to cisplatin sensitivity in NSCLC tumors were screened using P < 0.05 and foldchange > 2 as criteria. A total of 10 differentially expressed lncRNAs were obtained, and the upregulated lncRNAs included: FAM215B, CLLU1-AS1, SNHG9, MIR1915HG, CTB-61M7.1, LINC02694, LINC00525, MIF-AS1, and the downregulated lncRNAs included: GS1-98E2.1, GDF5-AS1, etc. Figure 2 shown.

[0030] The intersection of the differentially expressed genes in clinical samples and cell lines was used to obtain the upregulated lncRNA MIF-AS1 that was commonly expressed between the sensitive and resistant groups.

[0031] Since the increase in MIF-AS1 was large and had not been reported in the literature, RT-qPCR was used to further verify whether there was a difference in the expression of MIF-AS1 between the radiotherapy and chemotherapy sensitive group and the resistant group of NSCLC. The results of the RT-qPCR test showed that the expression of MIF-AS1 in the radiotherapy and chemotherapy resistant group of NSCLC was significantly increased compared with the radiotherapy and chemotherapy sensitive group (P<0.05). Figure 3 shown.

[0032] These results indicate that lncRNA MIF-AS1 has the potential to develop into a new biomarker for NSCLC.

[0033] 3.2 Predictive value of lncRNA MIF-AS1 for chemoradiotherapy sensitivity in NSCLC and its correlation with clinicopathological parameters in NSCLC patients

[0034] To explore the predictive value of lncRNA MIF-AS1 for the sensitivity of NSCLC patients to radiotherapy and chemotherapy, the ROC curve analysis was used to analyze the differences in the levels of lncRNA MIF-AS1 in the plasma of 61 NSCLC patients.

[0035] The results showed that the corresponding AUC value of lncRNA MIF-AS1 for distinguishing NSCLC patients who were insensitive to chemoradiotherapy from those who were sensitive to chemoradiotherapy was 0.775 (95% CI = 0.568 to 0.981, sensitivity = 81.8%, specificity = 80.0%), while the corresponding AUC value of the traditional biomarker CEA for distinguishing NSCLC patients who were insensitive to chemoradiotherapy from those who were sensitive to chemoradiotherapy was 0.590 (95% CI = 0.420 to 0.760, sensitivity = 81.8%, specificity = 48.0%). Figure 4 shown.

[0036] The results of this experiment showed that the predictive value of lncRNA MIF-AS1 for the sensitivity of NSCLC to radiotherapy and chemotherapy is better than the traditional biomarker CEA, and lncRNA MIF-AS1 can be used as a biomarker for the sensitivity of NSCLC to radiotherapy and chemotherapy.

[0037] To further explore the relationship between the expression of lncRNA MIF-AS1 and clinicopathological factors, the clinical data of the patients were collected and analyzed, and the results are shown in Table 1 .

[0038] Table 1 Correlation analysis between the expression of lncRNA MIF-AS1 in peripheral blood of NSCLC patients and clinicopathological parameters of NSCLC [median (interquartile range)]

[0039]

[0040] As can be seen, lncRNA MIF-AS1 expression was significantly correlated with tumor size (P < 0.05), but was not significantly associated with age, gender, smoking status, tumor stage, lymph node metastasis, or distant metastasis (P > 0.05). This suggests that lncRNA MIF-AS1, as a biomarker for NSCLC diagnosis, is less affected by interpatient variability and has promising application prospects.

[0041] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting the expression level of lncRNA MIF-AS1 in the preparation of a reagent for predicting the chemotherapy sensitivity of non-small cell lung cancer treated with cisplatin, wherein the sequence of the lncRNA MIF-AS1 is shown in SEQ ID NO.1.

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