Use of linc01978 inhibitors in the preparation of a medicament for treating colorectal cancer

Abstract

The application provides an application of an LINC01978 inhibitor in preparation of a drug for treating colorectal cancer, and belongs to the technical field of biological medicines. Experimental results show that after knocking out LINC01978, the proliferation, migration and invasion of colorectal cancer cells can be inhibited, and cell apoptosis can be promoted; after overexpressing LINC01978, the proliferation, migration and invasion of colorectal cancer cells can be promoted, and cell apoptosis can be reduced. It can be seen that the LINC01978 inhibitor has an effect of inhibiting the occurrence and development of colorectal cancer, has a potential therapeutic effect, and the LINC01978 inhibitor can be used for preparing the drug for treating colorectal cancer. The application first proposes and verifies that LINC01978 is a new gene therapy target point for colorectal cancer, has important significance for screening new drugs against colorectal cancer, and also provides a new idea for the treatment of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of LINC01978 inhibitors in the preparation of drugs for treating colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is a malignant tumor of the digestive system that seriously threatens human health, ranking third in incidence and second in mortality among malignant tumors. For patients diagnosed with CRC in its early stages, the 5-year survival rate after surgical treatment can reach 80-90%. However, because early symptoms of CRC are not obvious, about 50% of patients are already in the middle or late stages at the time of diagnosis, with the cancer having already metastasized. Surgical treatment combined with adjuvant radiotherapy, chemotherapy, and chemoradiotherapy has its limitations, significantly impacting treatment outcomes and resulting in a 5-year survival rate of less than 40%. Therefore, early diagnosis and treatment are crucial for reducing the mortality rate and improving the prognosis of colorectal cancer patients.

[0003] The development and progression of colorectal cancer is a complex process regulated by multiple genes. Whole-genome sequencing shows that at least 93% of the human genome sequence has transcriptomic activity, but only 2% of these sequences encode proteins; the remaining 98% are non-coding RNAs without protein-coding function. Among these, long non-coding RNAs (lncRNAs) are a class of non-coding RNA molecules with transcript lengths greater than 200 nt. Numerous studies have shown that lncRNAs are closely related to the development and progression of colorectal cancer, participating in biological processes such as proliferation, metastasis, invasion, and apoptosis. Research on lncRNAs in the field of colorectal cancer is still in its early stages and requires continuous exploration and investigation. Currently, the biological function of the LINC01978 gene in colorectal cancer has never been studied. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide the use of LINC01978 inhibitors in the preparation of drugs for treating colorectal cancer.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Application of LINC01978 inhibitors in the preparation of drugs for treating colorectal cancer.

[0006] Preferably, the LINC01978 inhibitor includes interfering molecules that inhibit the transcription and expression of LINC01978.

[0007] Preferably, the interfering molecules include shRNA, siRNA, dsRNA, miRNA, and antisense nucleic acid.

[0008] Preferably, the functional sequence of the shRNA is shown in SEQ ID NO:1.

[0009] Preferably, the LINC01978 inhibitor is used to: inhibit the proliferation of colorectal cancer cells; inhibit the migration of colorectal cancer cells; inhibit the invasion of colorectal cancer cells; or promote apoptosis of colorectal cancer cells.

[0010] Preferably, the drug comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a LINC01978 inhibitor.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of LINC01978 inhibitors in the preparation of drugs for treating colorectal cancer. Experimental results show that knocking out LINC01978 inhibits the proliferation, migration, and invasion of colorectal cancer cells and promotes apoptosis, while overexpression of LINC01978 promotes the proliferation, migration, and invasion of colorectal cancer cells and reduces apoptosis. Therefore, LINC01978 inhibitors have the effect of inhibiting the occurrence and development of colorectal cancer and possess potential therapeutic effects. LINC01978 inhibitors can be used to prepare drugs for treating colorectal cancer. This invention is the first to propose and verify LINC01978 as a novel gene therapy target for colorectal cancer, which is of great significance for screening new anti-colorectal cancer drugs and provides a new approach to the treatment of colorectal cancer. Attached Figure Description

[0012] Figure 1 Example 1: Microscopic observation of cell fluorescence carrying capacity; Figure 2 For comparison of mRNA expression levels in colorectal cancer cells transfected with LINC01978, note (****) P <0.0001 vs. OE-NC, ## P <0.01 vs. KO-NC, n=3); Figure 3 Results of colorectal cancer cell proliferation assay in each group; Figure 4 Results of colorectal cancer cell migration ability detection in each group; Figure 5 The results of the invasive ability test of colorectal cancer cells in each group; Figure 6 The results show the apoptosis ability of colorectal cancer cells in each group. Detailed Implementation

[0013] This invention provides the application of LINC01978 inhibitors in the preparation of drugs for treating colorectal cancer.

[0014] The sequence of the LINC01978 gene described in this invention is the NCBI sequence: 101928738.

[0015] In this invention, the LINC01978 inhibitor preferably includes an interfering molecule that inhibits the transcription and expression of LINC01978; the interfering molecule preferably includes shRNA, siRNA, dsRNA, miRNA, antisense nucleic acid, or a construct that can form shRNA, siRNA, dsRNA, miRNA, or antisense nucleic acid; the functional sequence of the shRNA is shown in SEQ ID NO:1: GATGCAGTGTTGTGCACAA, and the shRNA can be used to knock down the expression of LINC01978.

[0016] In this invention, the LINC01978 inhibitor is used to: inhibit the proliferation of colorectal cancer cells; inhibit the migration of colorectal cancer cells; inhibit the invasion of colorectal cancer cells; or promote apoptosis of colorectal cancer cells. Experimental results show that knocking out LINC01978 can inhibit the proliferation, migration, and invasion of colorectal cancer cells and promote apoptosis, while overexpression of LINC01978 can promote the proliferation, migration, and invasion of colorectal cancer cells and reduce apoptosis.

[0017] In this invention, the drug comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a LINC01978 inhibitor.

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1 Constructing LINC01978 overexpression and knockout cell models 1. Cell transfection (1) The LINC01978 overexpression vector was synthesized by Shanghai Jikai Gene Co., Ltd., and the negative control vector was LV4 (EF-1aF / GFP); (2) Design of LINC01978 shRNA sequence: GATGCAGTGTTGTGCACAA (SEQ ID NO:1). The LINC01978 knockdown vector was synthesized by Shanghai Jikai Gene Co., Ltd., and the negative control vector was LV-1 (pGLVU6 / GFP).

[0020] (3) Culture SW480 cells at 1×10 5 Inoculate 1 ml of medium into 12-well plates, adding 1 ml of culture medium (without antibiotics) to each well and incubate for 12 h.

[0021] (4) Perform cell transfection. Dilute the LINC01978 overexpression or knockout vector and negative control with 200 μl of OPTI-MEM medium, mix gently, and let stand at room temperature for 5 min.

[0022] (5) At the same time, dilute 5 μl of Lipofectamine 2000 with 200 μl of OPTI-MEM medium, mix gently, and let stand at room temperature for 5 min.

[0023] (6) Mix the diluted expression vector with Lipofectamine 2000, let it stand at room temperature for 25 min, and then add it to the cell culture wells of the corresponding group and shake well.

[0024] (7) After culturing the cells in a 37℃, 5% CO2 incubator for 24 h, the medium was changed. 48 h after transfection, the fluorescence carrying capacity of the cells was observed under a microscope. Figure 1 As shown.

[0025] Depend on Figure 1 It can be seen that the cells of the overexpression negative control (OE-NC group), the overexpression-LINC01978 (OE-LINC01978 group), the knockdown negative control (KO-NC group), and the knockdown-LINC01978 group (KO-LINC01978 group) all carried green fluorescence, which indicates that the LINC01978 overexpression vector and the knockdown vector have been successfully transfected into colorectal cancer cells.

[0026] Example 2 1. Extraction of total RNA from cells (1) Discard the culture medium of cells cultured in each group of well plates, wash three times with 1 ml PBS buffer, add 500 µl of Trizol reagent, pipette and aspirate to mix thoroughly, and let stand at room temperature for 5 min.

[0027] (2) Transfer the mixed liquid to a 1.5 ml centrifuge tube.

[0028] (3) Add 100 µl of chloroform to each centrifuge tube at a volume ratio of 1:5 with Trizol, tighten the cap, shake the centrifuge tube vigorously for 30 s, and then place it at room temperature for 5 min.

[0029] (4) Centrifuge at 4℃ and 12,000 g for 15 min. The mixture will be divided into three layers. The RNA will be dissolved in the upper transparent liquid phase. Transfer the supernatant to a new 1.5 ml centrifuge tube.

[0030] (5) Add an equal volume of isopropanol (pre-cooled to -20℃) to the centrifuge tube, tighten the cap, invert and mix several times, and then place at room temperature for 10 min.

[0031] (6) Centrifuge the centrifuge tube at 4°C and 12,000 g for 15 min and remove the supernatant.

[0032] (7) Add 500 µl of 75% ethanol (prepared with 0.1% DEPC-treated water) to the centrifuge tube, tighten the cap, shake for 15 s, and then place at room temperature for 5 min.

[0033] (8) Centrifuge the centrifuge tube at 4°C and 12,000 g for 15 min and remove the supernatant.

[0034] (9) Place the centrifuge tube in a clean laminar flow hood and air dry for 30 min. When there are no obvious water droplets in the centrifuge tube, add 10-15 μl of 0.1% DEPC-treated water to the centrifuge tube to dissolve the RNA.

[0035] (10) Use a micro spectrophotometer to detect RNA concentration and purity, and use agarose gel electrophoresis to detect RNA integrity. High-quality RNA can be used directly in the next experiment or stored at -80℃ for a long time.

[0036] 2. Reverse transcription The reverse transcription experiment was performed according to the reverse transcription kit instructions. The specific reverse transcription system is as follows: (1) Removal of genomic DNA Table 1 Composition of the reaction solution

[0037] Note: Prepare the reaction solution on ice according to the above method. Prepare the reaction mixture (excluding RNA) in advance according to the reaction number + 2, dispense it into each reaction tube, and finally add the RNA sample.

[0038] (2) The reaction tube was placed at room temperature and the reaction was carried out for 10 min.

[0039] (3) Reverse transcription PCR reaction Table 2 Reverse Transcription PCR Reaction Solution

[0040] (4) After mixing according to the above method, place it in a PCR instrument for reverse transcription under the following conditions: 37℃, 15 min, 85℃, 5 s, 4℃.

[0041] The above-described genomic DNA removal and reverse transcription reactions are reagents included in the TAKARA PrimeScript™ RT reagent Kit with gDNA Eraser. Simply follow the instructions on the kit.

[0042] 3. Quantitative Real-Time PCR (1) Primers Table 3 Primer sequences for real-time PCR

[0043] (2) Reaction system Table 4 Composition of the reaction system

[0044] (3) Reaction conditions Table 5 Reaction conditions

[0045] After the reaction, melting curve analysis was used to confirm the specificity of the PCR products and the presence of impurities and primer dimers. The Ct values ​​of each sample were obtained using Formula 2. -△△Ct The relative expression levels of mRNA in each sample were calculated using the method. Where ΔCt = target gene Ct value - internal reference gene Ct value, and -ΔCt = average ΔCt of the blank control group - ΔCt value of each sample group. See the detailed results below. Figure 2 .

[0046] Depend on Figure 2 The results showed that, compared with the OE-NC group, the mRNA expression level of LINC01978 in the OE-LINC01978 group was significantly increased; compared with the KO-NC group, the mRNA expression level of LINC01978 in the KO-LINC01978 group was significantly decreased. These results indicate that a differential expression system for LINC01978 in colorectal cancer cells has been established, and further experiments can be conducted based on this system.

[0047] (2) CCK8 assay to verify the effect of LINC01978 interference on the proliferation of colorectal cancer cells Five groups of cells were cultured in RPMI-1640 medium: SW480 cells (Control group), LINC01978 overexpression group (OE-LINC01978 group), LINC01978 overexpression negative control group (OE-Control group), LINC01978 knockdown group (KO-LINC01978 group), and LINC01978 knockdown negative control group (KO-Control group). Cells were cultured at a ratio of 5 × 10⁻⁶ cells / year. 3Cell suspensions were seeded into 96-well cell culture plates, and an appropriate amount of fresh culture medium was added. The plates were incubated overnight at 37°C with 5% CO2. After 48 h of cell culture, 20 μl of CCK-8 solution was added to each well, and the plates were incubated for another 3 h at 37°C with 5% CO2. The absorbance of each well was measured at 450 nm using a microplate reader. The results were then statistically analyzed. See attached table for details. Figure 3 .

[0048] Depend on Figure 3 It can be seen that, compared with the Control group, the cell proliferation capacity of the OE-LINC01978 group was significantly enhanced, the cell proliferation capacity of the KO-LINC01978 group was significantly reduced, and there was no significant difference in cell proliferation capacity between the OE-Control group and the KO-Control group.

[0049] (3) The scratch assay was used to verify the effect of interfering with LINC01978 on the migration of colorectal cancer cells. Five groups of cells were cultured in RPMI-1640 medium: SW480 cells (Control group), LINC01978 overexpression group (OE-LINC01978 group), LINC01978 overexpression negative control group (OE-Control group), LINC01978 knockdown group (KO-LINC01978 group), and LINC01978 knockdown negative control group (KO-Control group). Cells were cultured at a ratio of 5 × 10⁻⁶ cells / year. 4 Cell suspensions were seeded into 24-well cell culture plates, and an appropriate amount of fresh culture medium was added. The plates were incubated overnight at 37°C with 5% CO2. Cells were streaked vertically using a pipette tip, washed three times with PBS, and cultured again with serum-free medium. Images were taken under an inverted microscope, and the width of the streaks was recorded at 0 and 24 hours. Finally, the image data were collected and the experimental results were analyzed. Detailed results are shown in [link to results]. Figure 4 .

[0050] Depend on Figure 4 It was found that, when the scratch width was observed and recorded under a microscope at 40×, the cell migration ability of the OE-LINC01978 group was significantly enhanced compared with that of the Control group, while the cell migration ability of the KO-LINC01978 group was significantly reduced. No significant difference was found in the cell migration ability between the OE-Control group and the KO-Control group.

[0051] (4) Transwell assay to verify the effect of LINC01978 interference on the invasion of colorectal cancer cells. Matrigel, pre-chilled 24 h at 4°C, was diluted 1:3 with pre-chilled serum-free medium. 100 μl of the diluted Matrigel was added to each chamber and incubated until solidified. Five cell groups were cultured in RPMI-1640 medium: SW480 cells (Control group), LINC01978 overexpression group (OE-LINC01978 group), LINC01978 overexpression negative control group (OE-Control group), LINC01978 knockdown group (KO-LINC01978 group), and LINC01978 knockdown negative control group (KO-Control group). The cell concentration was adjusted to 5 × 10⁶ cells / mL with serum-free medium. 5 / ml, take 100 μl and drop it into the upper chamber of Transwell, add 500 μl of culture medium containing 10% FBS to the lower chamber. After routine incubation for 48 h, wipe the Matrigel gel in the lower chamber with a cotton swab, then fix with 4% paraformaldehyde, stain with crystal violet, observe under a microscope, photograph and count, and see the specific results in the following table. Figure 5 .

[0052] Depend on Figure 5 It can be seen that, compared with the Control group, the cell invasion ability of the OE-LINC01978 group was significantly enhanced, the cell invasion ability of the KO-LINC01978 group was significantly reduced, and there was no significant difference in cell invasion ability between the OE-Control group and the KO-Control group.

[0053] (5) Annexin V-FITC / PI double staining method to verify the effect of interfering with LINC01978 on apoptosis of colorectal cancer cells. Five groups of SW480 cells were cultured in RPMI-1640 medium: Control group, LINC01978 overexpression group (OE-LINC01978 group), LINC01978 overexpression negative control group (OE-Control group), LINC01978 knockdown group (KO-LINC01978 group), and LINC01978 knockdown negative control group (KO-Control group). 1×10⁶ cells were collected from each group. 5Cells were resuspended in 100 μl of 1×binding buffer according to the apoptosis kit instructions. 5 μl of FITC-annexin V and 10 μl of PI staining working solution were added. Cells were incubated at 37°C in the dark for 15 min. 400 μl of 1×annexin-binding buffer was added, mixed well, and placed on ice. Annexin V-FITC fluorescence selection was used for FITC pathway detection, and PI fluorescence selection was used for PI pathway detection. Cells from each group were sequentially analyzed by flow cytometry. Specific results are shown in [link to results]. Figure 6 .

[0054] Depend on Figure 6 It can be seen that, compared with the Control group, the apoptosis rate of cells in the OE-LINC01978 group was significantly reduced, while the apoptosis rate of cells in the KO-LINC01978 group was significantly increased. There was no significant difference in the apoptosis rate between the OE-Control group and the KO-Control group.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of LINC01978 inhibitors in the preparation of drugs for treating colorectal cancer, characterized in that, The LINC01978 inhibitor is an shRNA, and the functional sequence of the shRNA is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The LINC01978 inhibitor is used for: Inhibits the proliferation of colorectal cancer cells; Inhibits the migration of colorectal cancer cells; Inhibit the invasion of colorectal cancer cells; or Promotes apoptosis in colorectal cancer cells.

3. The application according to claim 1 or 2, characterized in that, The drug comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a LINC01978 inhibitor.

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