New application of piR-hsa-164586 and MYH9

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a novel application of piR-hsa-164586 and MYH9, the piR-hsa-164586 plays a role based on mutual combination of the piR-hsa-164586 and the MYH9, the piR-hsa-164586 regulates expression of the MYH9, plays a role in promoting NSCLC (non-small cell carcinoma) transfer, serves as a treatment target of NSCLC and becomes a potential treatment drug, the MYH9 is obtained through an RNApulldown experiment, and through protein mass spectrometry analysis, molecular docking and RIP experiment verification, the piR-hsa-164586 can be used for preparing the piR-hsa-164586 and the MYH9 have a good application prospect. The piR-hsa-164586 can positively regulate the expression of the MYH9, qRT-PCR, western blot and tissue immunofluorescence staining experiments are respectively carried out on a blank control group (NC), a knock-down piR-hsa-164586 group (KD) and an overexpression piR-hsa-164586 group (OE) to verify that the piR-hsa-164586 and the MYH9 are combined with each other, the expression of the MYH9 can be up-regulated in an NSCLC cell strain, and the verification experiment process is as follows: after the piR-hsa-164586 and the MYH9 are respectively knocked out, a Transwell experiment is carried out to verify that the piR-hsa-164586 and the MYH9 are respectively knocked out.

Description

Technical field:

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a new use of piR-hsa-164586 and MYH9, which promotes the progression of NSCLC (non-small cell lung cancer) by promoting A549 cell migration, and serves as a therapeutic target for NSCLC. Background technology:

[0002] The expression of piR-hsa-164586 in human serum exosomes is upregulated in serum exosomes and cells of NSCLC patients, which is of great value for the early diagnosis of lung cancer. In addition, piR-hsa-164586 is localized in the cell nucleus and can promote the proliferation, metastasis and angiogenesis of NSCLC cells, but inhibit their apoptosis. Based on this, the mechanism of action of piR-hsa-164586 may provide new ideas for the treatment of NSCLC. Non-coding small RNAs localized in the cell nucleus can participate in cancer progression by binding to proteins or RNA to form complexes. In order to further study the role of piR-hsa-164586 in NSCLC, proteins related to piR-hsa-164586 were first obtained by RNA pulldown experiments, and then protein mass spectrometry analysis, RIP experiments and molecular docking were performed. It was found and proved that a protein called MYH9 (myosin heavy chain 9) interacts with piR-hsa-164586 and is related to the occurrence of NSCLC.

[0003] MYH9 is involved in biological processes including cell migration, invasion, adhesion, cytokinesis and polarization, maintenance of cell shape and signal transduction. Its abnormal expression is closely related to the progression of various cancers. For example, MYH9-dependent polarization promotes colorectal cancer metastasis by accelerating focal adhesion assembly; MYH9 can enhance the progression of cancer stem cells in hepatocellular carcinoma; MYH9 affects the stem cell-like properties in NSCLC by activating AKT-mTOR signaling.

[0004] PIWI-interacting RNA (piRNA) belongs to a class of small non-coding RNA (ncRNA) with a length range of 24-31 nucleotides. It usually binds to the piwi protein family to play a regulatory role. PiRNA was originally found to play an important role in the reproductive system. Recent studies have shown that it is closely related to tumorigenesis. In addition, increasing evidence shows that abnormal expression of certain piRNAs may be involved in tumorigenesis and is associated with cancer prognosis. For example, piRNA-14633 can promote the progression of cervical cancer malignancies through MettL14-dependent m6A RNA methylation;

[0005] Abnormal expression of serum-derived piRNA-54265 can serve as a novel biomarker for detecting and diagnosing early human colorectal cancer.

[0006] When exploring the interaction between piRNA and MYH9: First, the proteins bound to piR-hsa-164586 were obtained through RNA pulldown experiments, and protein spectrum analysis was performed; then, the binding relationship between piR-hsa-164586 and the target protein MYH9 was verified through RNA binding protein immunoprecipitation (RIP) experiments, and the existence of binding sites between piR-hsa-164586 and the target protein MYH9 was proved through molecular docking experiments; finally, the regulatory effect of piR-hsa-164586 on downstream target proteins was verified based on mRNA levels and protein levels through qRT-PCR, protein immunoblotting and tissue immunofluorescence staining experiments, and the Transwell complementation experiment proved that MYH9 and piR-hsa-164586 can play a synergistic role in affecting the migration of A549 cells.

[0007] This shows that piR-hsa-164586 can positively regulate the expression of MYH9; piR-hsa-164586 can play a synergistic role with MYH9 to promote the progression of NSCLC by promoting the migration of A549 cells; piR-hsa-164586 and MYH9 can serve as potential therapeutic targets for NSCLC and have the potential to become new therapeutic drugs. Summary of the invention:

[0008] The novel use of piR-hsa-164586 and MYH9 involved in the present invention is based on piR-hsa-164586 promoting NSCLC metastasis by regulating the expression of MYH9, so as to serve as a therapeutic target for NSCLC.

[0009] The MYH9 involved in the present invention is obtained by RNA pulldown experiment and verified by protein mass spectrometry analysis, molecular docking and RIP experiment.

[0010] The piR-hsa-164586 involved in the present invention can positively regulate the expression of MYH9, which is verified by performing qRT-PCR, protein immunoblotting and tissue immunofluorescence staining experiments on a blank control group (NC), a piR-hsa-164586 knockdown group (KD) and a piR-hsa-164586 overexpression group (OE).

[0011] The piR-hsa-164586 and MYH9 involved in the present invention bind to each other and can upregulate the expression of MYH9 in NSCLC cell lines. The verification experiment is as follows:

[0012] After knocking out piR-hsa-164586 and MYH9, respectively, the number of NSCLC cell migration was significantly inhibited as confirmed by Transwell assay. Knocking down piR-hsa-164586 and MYH9 at the same time could further inhibit the number of A549 cell migration.

[0013] Overexpression of piR-hsa-164586 can significantly increase the number of A549 cell migration, while knocking down MYH9 significantly reduces the number of A549 cell migration;

[0014] These results suggest that the combination of piR-hsa-164586 and MYH9 can promote the migration of A549 cells and increase the metastatic ability of NSCLC.

[0015] Compared with the prior art, the present invention exerts its effects based on the mutual binding of piR-hsa-164586 and MYH9: piR-hsa-164586 regulates the expression of MYH9, plays a role in promoting the metastasis of NSCLC, and serves as a therapeutic target for NSCLC, becoming a potential therapeutic drug. Description of the drawings:

[0016] Figure 1 It is a representative peptide segment of the MYH9 protein analyzed by protein spectrum involved in the present invention.

[0017] Figure 2 This is a schematic diagram of WB of MYH9 protein pulled down by piR-hsa-164586 after the RNA pulldown operation involved in the present invention.

[0018] Figure 3 A schematic diagram of the interaction between piR-hsa-164586 and MYH9 shown by molecular docking involved in the present invention.

[0019] Figure 4 This is a schematic diagram of the qRT-PCR results of piR-hsa-164586 co-precipitated with MYH9 involved in the present invention, (n=3), *P<0.05.

[0020] Figure 5 This is a schematic diagram of the present invention's verification of the regulatory effect of piR-hsa-164586 on MYH9 at the mRNA level using qRT-PCR technology (n=3).

[0021] Figure 6The western blot diagram (A) and the western blot quantitative result diagram (n=3) (B) of the regulatory effect of piR-hsa-164586 on MYH9 based on protein level involved in the present invention.

[0022] Figure 7 Schematic diagram of immunofluorescence (×400 times, scalebar=50μm) (A) and quantitative analysis diagram of immunofluorescence (n=3) (B) of the regulatory effect of piR-hsa-164586 on MYH9 involved in the present invention, *P<0.05, ***P<0.001.

[0023] Figure 8 The results of the Transwell experiment (A) and the statistical data (B) of the effect of the binding between piR-hsa-164586 and MYH9 on cell migration involved in the present invention are shown (n=3), *P<0.05, **P<0.01; ns: no significance. Specific implementation method:

[0024] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] Embodiment 1:

[0026] The protein spectrum analysis involved in this embodiment is as follows Figure 1 The protein spectrum shown indicated that MYH9 included five peptides: QLLQANPILEAFGNAK, VASHLLGINVTDFTR, DLGEELEALKTELEDTLDSTAAQQELR, NTDQASMPDNTAAQK, and NAEQYKDQADK, which had the greatest binding potential with piR-hsa-164586 and were associated with lung cancer occurrence.

[0027] Embodiment 2:

[0028] The dissociation process of piR-hsa-164586 binding protein by RNA pulldown technology involved in this embodiment is as follows:

[0029] (1) Prepare two groups of cells (two 10 cm dishes / group), rinse with PBS (polybutylene succinate) 2-3 times, add 2 mL of cell lysis buffer to each dish, scrape the cells with a cell brush and transfer them to a 2 mL Ep tube (stainless steel electropolished tube), and use an automatic focused ultrasound sample processing system to lyse and disrupt the cells;

[0030] (2) Centrifuge at 12000 rpm for 40 min and take an appropriate amount of supernatant as input;

[0031] (3) Prepare the probe to a final concentration of 10 μM;

[0032] (4) performing magnetic separation;

[0033] (5) Add 1 mL of Buffer 1 and repeat step (4);

[0034] (6) Add 500 μL of biotinylated nucleic acid diluted with Buffer 1, vortex thoroughly to resuspend the magnetic beads, and mix the mixture thoroughly using a vortexer;

[0035] (7) Separate the mixed solution, transfer the supernatant, wash three times, and analyze the MYH9 protein pulled down by piR-hsa-164586 by western blot after RNA pulldown. Figure 2 shown.

[0036] Embodiment 3:

[0037] The process of proving the mutual binding of piR-hsa-164586 and MYH9 by molecular docking in this embodiment is as follows:

[0038] (1) Predict the secondary structure of piRNA-hsa-164536 using Vflod2D;

[0039] (2) Predict the tertiary structure of piRNA-hsa-164536 using Vflod3D;

[0040] (3) Molecular docking was performed using the GRAMM website. The results are as follows Figure 3 shown.

[0041] Embodiment 4:

[0042] The process of RNA binding protein aggregation and testing by RNA binding protein immunoprecipitation (RIP) in this embodiment is as follows:

[0043] (1) Add Buffer A working solution to the cells and lyse them on ice. Vortex and mix every 10 min.

[0044] (2) Centrifugation (4°C, 14,000 g, 10 min) and retaining the supernatant;

[0045] (3) Take 100 μL of the supernatant as input;

[0046] (4) Add magnetic beads to the remaining supernatant and rotate evenly;

[0047] (5) separating the supernatant from the magnetic beads and retaining the supernatant;

[0048] (6) Mix the magnetic beads, the antibody of the target protein, the internal reference (IgG), and Buffer A separately, and rotate and mix at 4°C for 2 h;

[0049] (7) magnetic separation to remove the supernatant;

[0050] (8) washing, magnetic separation, and removing the supernatant;

[0051] (9) Add 1× Buffer A and cell lysate to the mixed magnetic beads (containing the target antibody and IgG) and rotate at a low speed (10-15 rpm) in a refrigerator at 4°C overnight;

[0052] (10) Magnetic separation: retain the magnetic beads, add 1 mL of 1× Buffer B to the mixed magnetic beads, mix well, separate, and repeat the operation 5 times;

[0053] (11) Add Buffer E to the mixed magnetic beads of different groups (including the previously reserved Input tube), vortex and mix, perform magnetic separation, and retain the supernatant;

[0054] (12) Remove DNA using a DR Columns filter (14,000 g, centrifugation for 2 min), collect the filtrate, add 70% ethanol (equal volume to the filtrate), and mix by pipetting;

[0055] (13) Filter the purified RNA in an RC Column (12000 g, centrifuge for 30-60 s), add 500 μL of Buffer F (containing ethanol) and L Buffer G (containing ethanol) to the RC Column respectively, and centrifuge (12000 g, 2 min / time) 3 times, removing the filtrate each time; add an appropriate amount of RNase-free water to the center of the membrane of the new RNase-free centrifuge tube; dissolve at room temperature for 2 min; collect RNA (12000 g, centrifuge for 1 min);

[0056] (14) The product was verified by qRT-PCR, and the results were as follows Figure 4 shown.

[0057] Embodiment 5:

[0058] The process involved in this example of verifying the regulatory effect of piR-hsa-164586 on downstream target proteins based on mRNA levels by qRT-PCR is as follows:

[0059] (1) Design MYH9-related primers;

[0060] (2) Verified by qRT-PCR, the results are as follows Figure 5 shown.

[0061] Embodiment 6:

[0062] The process of verifying the regulatory effect of piR-hsa-164586 on downstream target proteins based on protein level by protein immunoblotting in this example is as follows:

[0063] (1) Add an appropriate amount of RIPA (containing 1% PMSF) lysis buffer to the extracted and purified exosomes and lyse them on ice for about 30 min to obtain a sample;

[0064] (2) Centrifuge the sample in a centrifuge at 4°C (12,000 rpm, 15 min);

[0065] (3) Take the supernatant, use the protein supernatant to make 5→1 loading buffer, place in a 95℃ metal bath for 5 min, and use it for electrophoresis after cooling;

[0066] (4) First, clean the 1.0 mm WB special glass plate with detergent, then rinse it with tap water, and finally rinse it with double distilled water and dry it;

[0067] (5) Fill with double distilled water and observe for 5 minutes. If the liquid level does not drop significantly, proceed with the subsequent experiment;

[0068] (6) Add 5-6 ml of lower layer glue;

[0069] (7) Add anhydrous ethanol until the liquid level is flattened, and leave at room temperature for about 15 min. After the lower layer of glue solidifies, pour out the anhydrous ethanol;

[0070] (8) Prepare a concentrated gel and place it in a suitable comb. After the lower layer of gel solidifies, slowly pull out the comb and pour 1 protein electrophoresis buffer into the electrophoresis tank for subsequent electrophoresis;

[0071] (9) Load the protein sample and run at 80V for 30 min. When the sample is aligned with the boundary between the upper and lower layers, switch to 120V and run for 90 min until the electrophoresis is completed.

[0072] (10) Cutting the PVDF membrane to match the size of the gel;

[0073] (11) Activate the PVDF membrane with methanol and fit it tightly to the gel surface;

[0074] (12) placing the transfer device in the transfer tank, adding an ice box to the transfer tank and then placing it in a foam box filled with ice;

[0075] (13) Transfer the membrane at a constant current of 300 mA, and the transfer time (min) is approximately equal to the protein molecular weight;

[0076] (14) Block the transferred PVDF membrane with a rapid blocking solution at 25 rpm at room temperature for 20 min;

[0077] (15) Place the PVDF membrane in the primary antibody dilution solution and incubate it at room temperature for 2 h on a shaker at 25 rpm or in a refrigerator at 4 °C overnight (12 h);

[0078] (16) Wash the membrane with 1× TBST at 50 rpm for 10 min each time, for a total of three times;

[0079] (17) Prepare 5% skim milk powder in 1× TBST, dilute the secondary antibody, and incubate at room temperature for 1.5 h;

[0080] (18) Repeat step (3);

[0081] (19) After the membrane is completely drained of water, it is immersed in a chemiluminescent solution and placed in a chemiluminescent instrument for exposure and development. The results are as follows: Figure 6 shown.

[0082] Embodiment 7:

[0083] The process of verifying the regulatory effect of piR-hsa-164586 on downstream target proteins based on protein level by immunofluorescence technology in this embodiment is as follows:

[0084] (1) Preparation of tissue frozen sections;

[0085] (2) Fix tissue frozen sections with 4% paraformaldehyde for 20 min;

[0086] (3) Rinse tissue frozen sections three times with PBS buffer;

[0087] (4) Block tissue frozen sections with antibody blocking solution at room temperature for 1 h;

[0088] (5) Incubate with primary antibody at 4°C in the dark overnight;

[0089] (6) Rinse the primary antibody 2-3 times with PBS, each time for 3-5 minutes;

[0090] (7) Incubate the secondary antibody at room temperature for 2 h;

[0091] (8) Wash off the secondary antibody and use DAPI to stain the cell nucleus;

[0092] (9) Observe and acquire images under an inverted fluorescence microscope;

[0093] (10) Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, USA). Figure 7As shown, the fluorescence signal (red) of MYH9 was significantly enhanced after piR-hsa-164586 was added (P < 0.05), and the fluorescence signal of MYH9 was significantly weakened after knocking down piR-hsa-164586 (P < 0.05), indicating that piR-hsa-164586 has a positive regulatory effect on MYH9.

[0094] Embodiment 8:

[0095] The process of comparing the migration ability of different groups of cells through Transwell experiment in this embodiment is as follows:

[0096] (1) The NSCLC cell groups were divided into: NC group, piR-hsa-164586 overexpression group, piR-hsa-164586 knockdown group, MYH9 knockdown group, piR-hsa-164586 overexpression group +

[0097] MYH9 knockdown group, piR-hsa-164586 knockdown group + MYH9 knockdown group;

[0098] (2) 1×10 per hole 5 Cells were seeded in 12-well plates, and after overnight culture, the nucleic acid analogs and inhibitors of piR-hsa-164586 were transfected into the cells;

[0099] (3) 24 h after transfection, the cells were digested and centrifuged, and the cell suspension (resuspended in serum-free medium) was added to the upper chamber, and DMEM medium containing 15% FBS was added to the lower chamber, and the cells were cultured in a CO2 incubator at 37°C for 24 h;

[0100] (4) Aspirate the old culture medium in the upper chamber, rinse, and add formaldehyde (4%) to fix for 20 min;

[0101] (5) Wash three times with PBS buffer;

[0102] (6) Stain with 0.1% crystal violet solution for 10 min, wash with PBS buffer and observe the acquired image under a microscope. Figure 8 As shown,

[0103] When MYH9 was knocked down, the number of A549 cells that migrated was reduced (P < 0.05), and knocking down MYH9 and piR-hsa-164586 at the same time aggravated this inhibitory effect;

[0104] When piR-hsa-164586 was overexpressed, the number of migrating cells increased significantly, and knocking down MYH9 significantly inhibited this migration effect (P < 0.05);

[0105] This indicates that after piR-hsa-164586 and MYH9 bind to each other, they play a regulatory role in the migration ability of A549 cells.

[0106] Among them, the statistical methods involved in Examples 3-8 used GraphPad Prism 8.0 software (GraphPad Software, USA) and SPSS 22.0 software (IBM, Germany) to analyze the experimental data, and the normality test of data distribution was evaluated by Kolmoorov-smirnov. If the data conformed to the normal distribution, the t-test was selected for analysis between the two groups of data;

[0107] If it does not conform to normal distribution, Wilcoxontest is selected for analysis;

[0108] ANOVA analysis was selected for data analysis of more than two groups;

[0109] Image processing was performed and analyzed using ImageJ (V1.8.0.112), and ROC curves were constructed to evaluate the diagnostic performance, with P values ​​< 0.05 considered statistically significant;

[0110] All tests were two-tailed.

Claims

1. A new use of piR-hsa-164586 and MYH9, characterized in that: As a therapeutic target for NSCLC.

2. The novel use of piR-hsa-164586 and MYH9 according to claim 1, characterized in that The principle is that piR-hsa-164586 promotes NSCLC metastasis by regulating the expression of MYH9.

3. The novel use of piR-hsa-164586 and MYH9 according to claim 1 or 2, characterized in that MYH9 was obtained by RNA pulldown experiments and verified by protein mass spectrometry analysis, molecular docking and RIP experiments.

4. The novel use of piR-hsa-164586 and MYH9 according to claim 1 or 2, characterized in that piR-hsa-164586 positively regulates the expression of MYH9, which was verified by qRT-PCR, protein immunoblotting and tissue immunofluorescence staining experiments in the blank control group, piR-hsa-164586 knockdown group and piR-hsa-164586 overexpression group.

5. The novel use of piR-hsa-164586 and MYH9 according to claim 1 or 2, characterized in that piR-hsa-164586 and MYH9 bind to each other and upregulate the expression of MYH9 in NSCLC cell lines. The verification experiment is as follows: After knocking out piR-hsa-164586 and MYH9, respectively, the number of NSCLC cell migration was inhibited as confirmed by Transwell assay, and knocking down piR-hsa-164586 and MYH9 further inhibited the number of A549 cell migration; Overexpression of piR-hsa-164586 increased the number of migrating A549 cells, while knockdown of MYH9 decreased the number of migrating A549 cells; These results suggest that the combination of piR-hsa-164586 and MYH9 can promote the migration of A549 cells and increase the metastatic ability of NSCLC.

Images