Application of HHIPL2-HNRNPC signaling pathway in the treatment of non-small cell lung cancer and preparation of targeted drugs

Abstract

The present invention relates to the application of HHIPL2-HNRNPC signaling pathway in the treatment of non-small cell lung cancer and the preparation of targeted drugs. The HHIPL2-HNRNPC signaling pathway includes HHIPL2 gene and HNRNPC gene, and the nucleotide sequences are shown in SEQ ID NO.1~2 respectively. The present invention finds that targeted interference with the activity of HHIPL2-HNRNPC signaling pathway can more effectively inhibit the malignant progression of non-small cell lung cancer than inhibiting the expression level of HHIPL2 or HNRNPC gene alone. The designed siMIX can specifically interfere with the mRNA levels of HHIPL2 and HNRNPC at the same time, showing a more effective anti-proliferation and metastasis effect of non-small cell lung cancer. Therefore, the HHIPL2-HNRNPC signaling pathway can be used as a drug target and applied to the research and development and preparation of targeted drugs for the treatment of non-small cell lung cancer.

Description

Technical Field

[0001] The invention relates to application of HHIPL2-HNRNPC signaling pathway in treating non-small cell lung cancer and preparing targeted drugs, and belongs to the technical field of biomedicine. Background Art

[0002] Lung cancer has become the most common human malignancy worldwide and ranks first among the causes of cancer-related deaths. In my country, lung cancer ranks first in the incidence of cancer in the male population and second in the incidence of cancer in the female population, second only to breast cancer. Lung cancer is also the leading cause of cancer-related deaths in my country. About 85% of lung cancer pathological types are non-small cell lung cancer (NSCLC), which includes lung adenocarcinoma, lung squamous cell carcinoma and lung large cell carcinoma. Existing targeted drugs mainly target EGFR sensitive mutations, and drug resistance often occurs during the treatment of NSCLC. Nearly half of NSCLC patients do not have EGFR sensitive mutations, which seriously limits the application and efficacy of targeted drugs in the clinical treatment of NSCLC. Therefore, exploring new targets and formulating corresponding intervention strategies are urgent issues to be addressed in the targeted treatment of NSCLC.

[0003] siRNA is a double-stranded RNA composed of 19-25 pairs of nucleotides. It was first discovered in plants by David Baulcombe's team. In 2001, Thomas Tuschl's team first discovered the mRNA interference effect of siRNA in mammals, thus opening up the wide application of siRNA in biomedical research and drug development. Ensuring the specificity and effectiveness of knocking down the target gene mRNA is the key to RNA interference technology and an important reference for evaluating the success of siRNA design. Therefore, designing siRNA with high knockdown efficiency and high specificity for the target gene will provide important technical support for the functional verification of new driver genes in NSCLC and the formulation of targeted intervention strategies. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides the application of the HHIPL2-HNRNPC signaling pathway in the treatment of non-small cell lung cancer and the preparation of targeted drugs.

[0005] The technical solution of the present invention is as follows:

[0006] Application of HHIPL2-HNRNPC signaling pathway as a drug target in the preparation of targeted drugs for the treatment of non-small cell lung cancer.

[0007] Preferably according to the present invention, the HHIPL2-HNRNPC signaling pathway includes the HHIPL2 gene and the HNRNPC gene.

[0008] Preferably according to the present invention, the mRNA nucleotide sequence of the HHIPL2 gene is shown as SEQ ID NO.1, and the mRNA nucleotide sequence of the HNRNPC gene is shown as SEQ ID NO.2.

[0009] Preferably according to the present invention, the targeted drug for treating non-small cell lung cancer takes HHIPL2 and HNRNPC genes as intervention targets and is based on interfering with HHIPL2 and HNRNPC mRNA, and can efficiently and specifically inhibit the expression levels of HHIPL2 and HNRNPC genes.

[0010] Preferably according to the present invention, the targeted drug for treating non-small cell lung cancer is siRNA, shRNA, microRNA or HHIPL2 and HNRNPC mRNA chemical inhibitor.

[0011] Application of siRNA that specifically interferes with HHIPL2 gene and siRNA that specifically interferes with HNRNPC gene in the preparation of targeted drugs for the treatment of non-small cell lung cancer.

[0012] Preferably according to the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HHIPL2 gene is shown as SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC gene is shown as SEQ ID NO.4.

[0013] Preferably, according to the present invention, the molar ratio of the siRNA that specifically interferes with the HHIPL2 gene to the siRNA that specifically interferes with the HNRNPC gene in the targeted drug for treating non-small cell lung cancer is 1:1.

[0014] A targeted drug for treating non-small cell lung cancer comprises siRNA that specifically interferes with HHIPL2 gene expression and siRNA that specifically interferes with HNRNPC gene expression.

[0015] Preferably according to the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HHIPL2 gene is shown as SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC gene is shown as SEQ ID NO.4.

[0016] Beneficial effects:

[0017] The present invention discovers for the first time that targeted interference with the activity of the HHIPL2-HNRNPC signaling pathway can more effectively inhibit the malignant progression of non-small cell lung cancer than inhibiting the expression level of HHIPL2 or HNRNPC genes alone. The siMIX designed by the present invention can specifically interfere with the mRNA levels of HHIPL2 and HNRNPC at the same time. When the same dose of siRNA is transfected, cell function experiments show that siMIX shows a more effective anti-proliferation and anti-metastasis effect on non-small cell lung cancer compared to siHHIPL2 or siHNRNPC. Therefore, the HHIPL2-HNRNPC signaling pathway can be used as a drug target for the development and preparation of targeted drugs for the treatment of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A549 cells were selected and transfected with the same dose of siMIX or siNC. Reverse transcription-real-time fluorescence quantitative PCR was used to detect the mRNA levels of HHIPL2 and HNRNPC. The statistical significance of the differences among the groups was determined by Student's t test.

[0019] Figure 2 siHHIPL2, siHNRNPC, siMIX or siNC were transfected into A549 cells respectively. The transfection dose of each siRNA was the same. The proliferation ability of A549 cells was detected by CCK-8 assay, and the statistical significance of the differences among the groups was verified by two-way ANOVA test.

[0020] Figure 3 A549 cells were selected and transfected with the same dose of siHHIPL2, siHNRNPC, siMIX or siNC, and the migration and invasion abilities of A549 cells were detected by Transwell assay. The statistical significance of the differences among the groups was determined by one-way ANOVA. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with specific experimental examples, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the examples are all common commercially available products unless otherwise specified.

[0022] Human non-small cell lung cancer cell line A549 cells are available from Shanghai Fuheng Biotechnology Co., Ltd.

[0023] siHHIPL2 targeting sequence: 5′-cacaatcgcaagttctata-3′;

[0024] siHNRNPC targeting sequence: 5′-gaaggagctgacccagata-3′.

[0025] Example 1

[0026] The inventors' previous research found that the HHIPL2-HNRNPC signaling pathway was abnormally activated in non-small cell lung cancer, and then further studied its mechanism and found that the HHIPL2-HNRNPC pathway plays an important role in the proliferation and metastasis of cancer cells. Therefore, the inventors believe that inhibiting the HHIPL2-HNRNPC signaling pathway will point out a new direction for the formulation of targeted treatment strategies for non-small cell lung cancer.

[0027] Example 2

[0028] The nucleotide sequences of HHIPL2 and HNRNPC mRNA were obtained through the NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, and siRNAs (siHHIPL2 and siHNRNPC) that specifically interfere with the expression levels of HHIPL2 and HNRNPC mRNA were designed, respectively, and their target sequences are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The negative control of the above siRNAs is siNC, which is a conventional existing sequence. siHHIPL2 and siHNRNPC were mixed in a molar ratio of 1:1 to obtain siMIX. The same dose (volume 3μL, concentration 10μM) of siNC and siMIX were transfected into A549 cells, RNA was extracted after 48 hours, and reverse transcription-real-time fluorescence quantitative PCR was used to detect the levels of HHIPL2 and HNRNPC mRNA. The specific results are shown as follows Figure 1 shown.

[0029] Depend on Figure 1 It can be seen that compared with transfection of siNC, transfection of siMIX can significantly downregulate the expression levels of HHIPL2 and HNRNPC mRNA in A549 cells.

[0030] The specific implementation process is as follows:

[0031] (1) A549 cells were plated in a 6-well plate. When the cell growth density reached about 60%, the same dose (volume 3 μL, concentration 10 μM) of siMIX and siNC were transfected into A549 cells using the transfection reagent Lipofectamine RNAiMAX Reagent (Life technologies, 13778-150).

[0032] (2) The cells were collected 48 hours after transfection, and RNA was extracted using the RNA-Quick Purification Kit (Shanghai Yishan, RN001). The concentration of the extracted RNA was detected using NanoDrop2000, and the integrity of the extracted RNA was evaluated by agarose gel electrophoresis;

[0033] (3) Using the extracted RNA as a template, reverse transcription kit LunaScript TM RT SuperMix Kit (NEB, E3010) was used for reverse transcription to synthesize cDNA products. Green Master Mix (ThermoFisher Scientific, 4367659) kit and QuantStudio TM 5System (Thermo Fisher Scientific) PCR instrument was used for fluorescence quantitative PCR, with ACTB mRNA level as the internal reference, according to 2 -ΔΔCT The relative expression levels of HHIPL2 and HNRNPC mRNA were calculated by the formula.

[0034] Example 3

[0035] A549 cells were transfected with siNC, siHHIPL2, siHNRNPC and siMIX respectively, with the same transfection dose (volume 3 μL, concentration 10 μM). CCK-8 assay was used to detect the changes in A549 cell viability. The specific results are as follows: Figure 2 shown.

[0036] Depend on Figure 2 It can be seen that the proliferation ability of A549 cells transfected with siHHIPL2, siHNRNPC or siMIX was significantly weakened compared with the control group cells, but siMIX showed a more significant cell proliferation inhibitory effect compared with the same dose of siHHIPL2 or siHNRNPC.

[0037] The specific implementation process is as follows:

[0038] (1) The same dose (volume 3 μL, concentration 10 μM) of siNC, siHHIPL2, siHNRNPC and siMIX were selected and transfected into A549 cells respectively. The specific method is detailed in Example 2. The transfected cells were collected after 48 hours, and the different transfected cells were re-cultured in 96-well plates (2,000 cells / well) and divided into 4 groups (Day 0, 1, 2, 3). Each group included siNC, siHHIPL2, siHNRNPC and siMIX transfected cells, and each cell was plated in 3 replicate wells;

[0039] (2) After the cells adhere to the wall (about 2-4 hours), add CCK-8 reagent (TargetMol, C0005) to the Day 0 group, 10 μL per well, place in a 37°C incubator for 1 hour, shake briefly, place in a microplate reader and measure the absorbance at 450 nm. This is the absorbance value of the initial seeded cells (Day 0);

[0040] (3) After completing the initial cell absorbance measurement, measure the absorbance values ​​of Day 1, 2, and 3 groups every 24 hours, and calculate the relative change level of cell viability in each group with Day 0 group as a reference.

[0041] Example 4

[0042] A549 cells were transfected with siNC, siHHIPL2, siHNRNPC or siMIX. The siRNA dose was the same (volume 3 μL, concentration 10 μM). Transwell assay was used to detect cell migration and invasion ability. The specific results are shown in Figure 3 shown.

[0043] Depend on Figure 3 It can be seen that under the condition of transfection with the same dose of siRNAs, A549 cells transfected with siMIX showed weaker cell migration and invasion ability than those transfected with siHHIPL2 or siHNRNPC, indicating that simultaneous interference with the expression levels of HHIPL2 and HNRNPC genes has a more significant anti-cancer effect than knocking down the expression of either gene alone.

[0044] The specific implementation process is as follows:

[0045] (1) A549 cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of siNC, siHHIPL2, siHNRNPC and siMIX, respectively, using the method described in Example 2;

[0046] (2) Transwell chambers (Corning, 3422) were selected. The chambers pre-coated with Matrigel were used to detect cell invasion ability, and the chambers without Matrigel were used to evaluate cell migration. The Transwell chambers were placed in 24-well plates (600 μL complete culture medium was pre-added to each well);

[0047] (3) 48 hours after transfection, the cell pellet was collected and resuspended in serum-free medium to adjust the cell concentration to 100,000 cells / mL. 200 μL of the cell suspension was added to the Transwell chamber and the 24-well plate was placed in a cell incubator (37°C, 5% CO2) for 48 hours.

[0048] (4) Remove the Transwell chamber and discard the culture medium. Fix the cells with 4% paraformaldehyde at room temperature for 15 minutes and then stain them with 0.1% crystal violet overnight. Use a cotton swab to scrape off the cells that have not migrated or invaded the inner side of the chamber. Observe the cells that have migrated or invaded to the outer side of the chamber under an inverted microscope and take photos to record.

Claims

1. Use of siRNA that specifically interferes with HHIPL2 gene expression and siRNA that specifically interferes with HNRNPC gene expression in the preparation of targeted drugs for the treatment of non-small cell lung cancer, characterized in that: The targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HHIPL2 gene is shown in SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the HNRNPC gene is shown in SEQ ID NO.4; the mRNA nucleotide sequence of the HHIPL2 gene is shown in SEQ ID NO.1, and the mRNA nucleotide sequence of the HNRNPC gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The molar ratio of the siRNA that specifically interferes with the expression of the HHIPL2 gene and the siRNA that specifically interferes with the expression of the HNRNPC gene in the targeted drug for treating non-small cell lung cancer is 1:

1.

3. A targeted drug for treating non-small cell lung cancer, characterized in that: The targeted drug for treating non-small cell lung cancer comprises siRNA that specifically interferes with the expression of HHIPL2 gene and siRNA that specifically interferes with the expression of HNRNPC gene; The targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HHIPL2 gene is shown in SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the HNRNPC gene is shown in SEQ ID NO.4.

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