Application of miR-491-5p in sepsis complicated with acute lung injury

Abstract

The invention discloses application of miR-491-5p in sepsis complicated with acute lung injury. The research finds that the miR-491-5p is highly expressed in peripheral blood of a sepsis patient and peripheral blood of a cecum ligation perforation model mouse; the reagent for inhibiting the expression of the miR-491-5p in the hPAEC cell inhibits the expression of pro-inflammatory genes IL-1beta, TNF-alpha, IL-6, CXCL10 and CRP of the hPAEC cell, inhibits the secretion of inflammatory factors IL-1beta, TNF-alpha and IL-6 of the hPAEC cell, promotes the proliferation activity of the hPAEC cell, promotes the migration activity of the hPAEC cell, inhibits the apoptosis of the hPAEC cell, and inhibits the expression of the miR-491-5p in the hPAEC cell. The inhibition of miR-491-5p expression provides a new possible treatment means for the treatment of sepsis complicated with acute lung injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of miR-491-5p in sepsis-induced acute lung injury. Background Art

[0002] Sepsis refers to life-threatening organ dysfunction caused by an imbalanced host response to infection. It is a common and serious clinical disease with a high mortality rate. Although the 2021 "Surviving Sepsis Campaign" recommended personalized treatment strategies and care methods for sepsis patients, the mortality rate of sepsis patients remains high, with approximately 48.9 million cases of sepsis and 11 million sepsis-related deaths worldwide each year. [1] .

[0003] Acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is a common clinical complication of sepsis. It is the result of the accumulation of protein-rich inflammatory edema fluid in the alveolar cavity due to damage to the alveolar epithelium and pulmonary endothelium. Compared with ALI / ARDS caused by other reasons, sepsis-related ALI / ARDS has a higher mortality rate of 30% to 40%, mainly manifested by diffuse alveolar damage, hypoxemia and respiratory distress, which seriously endangers human health and has become a huge public health burden. Its pathological changes mainly include three stages: exudative stage, recovery stage and fibrosis stage. At present, the pathogenesis is not very clear, and the diagnosis is only based on the consensus syndrome standard. There is still a lack of specific drugs and specific treatment measures for treatment. The current treatment focus is still to rely on lung protective ventilation, prone ventilation and fluid management to relieve symptoms. Even patients who survive ALI / ARDS may suffer from persistent functional and / or psychological sequelae, including motor limitation, psychological sequelae, decreased quality of life, and economic burden. [1] An epidemiological survey included 2322 patients with sepsis in 44 intensive care units in my country. The results showed that 68.2% of sepsis patients had ALI, and the 90-day mortality rate was 35.5%. [2] .

[0004] It has been found that miR-491-5p has the following functions: targeting FOLR1 to inhibit breast cancer cell proliferation and invasion, which may be related to changing the expression of proteins related to the AKT / mTOR signaling pathway [3] ; Regulates proliferation and apoptosis of tongue squamous cell carcinoma cells [4] ; Inhibits hepatoblastoma cell proliferation and migration by negatively regulating FGFR4, which may be related to the inactivation of GSK3β / β-catenin signaling pathway [5] And other functions.

[0005] Therefore, it is urgent to further clarify the pathogenesis of sepsis combined with acute lung injury and actively seek new targets and methods for the treatment of ALI.

[0006] References:

[0007] [1]Xue Yanhong, Yang Xiufen. Pathogenesis and treatment progress of acute lung injury in sepsis[J]. Journal of Hebei Medical University, 2024, (Issue 4).

[0008] [2] Liu Yumo 1 , Pan Guo Hairong 1 , Liang Qun 1,2 .Research progress on the pathogenesis of acute lung injury in sepsis[J]. Journal of Difficult Diseases, 2022, (Issue 8).

[0009] [3] Zhu Chao, Zhang Ligong, Guo Chenxu, Yin Faxiang, Xu Rui, Qian Jun. Effects of miR-491-5p targeting FOLR1 on proliferation and invasion of breast cancer cells[J]. Journal of Shanxi Medical University, 2023, (Issue 10).

[0010] [4] Tang Danping, Cheng Zhigang, Deng Shaolin. Effects of miR-491-5p targeting CCL22 on proliferation and apoptosis of tongue squamous cell carcinoma cells[J]. Chinese Journal of Gerontology, 2021, (Issue 19).

[0011] [5] Zhao Tianjiao 1 , Geng Jianlei 2 ,Wen Chan 3 , Ren Hui 2 ,Liu Feng 2 ,Wei Ping 4 .miR-491-5p targets FGFR4 to regulate proliferation and migration of hepatoblastoma cells[J]. Chinese Journal of Cancer Prevention and Treatment, 2021, (Issue 1). Summary of the invention

[0012] In view of the high mortality rate of patients with sepsis combined with acute lung injury and limited treatment methods, the present application study found that inhibiting miR-491-5p expression can inhibit the expression of inflammatory genes in hPAEC cells, inhibit the secretion of inflammatory factors, promote hPAEC cell proliferation, inhibit hPAEC cell migration, and inhibit hPAEC apoptosis, providing a new possible treatment option for patients with sepsis combined with acute lung injury; high expression of miR-491-5p can promote the level of inflammatory factors, promote the secretion of inflammatory factors, inhibit hPAEC cell proliferation, promote hPAEC cell migration, and promote hPAEC apoptosis. High expression of miR-491-5p can provide a new possible method for constructing a sepsis combined with acute lung injury model. To achieve the above purpose, the specific technical scheme of the present invention is as follows:

[0013] First, investigate the expression of miR-491-5p in the peripheral blood of patients with sepsis

[0014] Peripheral blood samples were collected from healthy subjects (n=30) and patients with sepsis (n=30), and total RNA was extracted using Trizol Reagent, and RNA quality was tested and quantified. RT-qPCR was used to detect the expression level of miR-491-5p.

[0015] Results: There was a significant difference in the expression of miR-491-5p in peripheral blood samples between patients with sepsis and normal controls, and miR-491-5p was highly expressed in patients with sepsis.

[0016] Second, investigate the expression of miR-491-5p in the peripheral blood of mice with cecal ligation and puncture model

[0017] The cecal ligation and puncture (CLP) model of C57BL / 6J black mice was established. Total RNA was extracted from peripheral blood samples, lung tissues, and kidney tissues of cecal ligation and puncture model mice and normal mice, and the expression level of miR-491-5p was detected by RT-qPCR.

[0018] Results: There were significant differences in the expression of miR-491-5p in peripheral blood samples, lung tissues, and kidney tissues between cecal ligation and puncture model mice and normal mice, with P values ​​less than 0.0001. miR-491-5p was highly expressed in peripheral blood samples, lung tissues, and kidney tissues of cecal ligation and puncture model mice.

[0019] The third aspect is to investigate the effect of miR-491-5p knockdown on inflammatory factor levels and pulmonary artery endothelial cells

[0020] (I) Effect of miR-491-5p knockdown on pro-inflammatory gene expression

[0021] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. M1 induction was performed 48 hours later, and total RNA was extracted. RT-qPCR was used to detect the transcriptional expression levels of pro-inflammatory genes IL-1β, TNF-α, IL-6, CXCL-10, and CRP in hPAEC cells.

[0022] Conclusion: Knockdown of miR-491-5p inhibited the expression of pro-inflammatory genes in hPAEC cells.

[0023] (II) Effect of miR-491-5p knockdown on the secretion of inflammatory factors

[0024] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. The supernatant of hPAEC cells was collected and the secretion levels of inflammatory factors IL-1β, TNF-α, and IL-6 were detected by ELISA.

[0025] Conclusion: Knockdown of miR-491-5p inhibits the secretion of inflammatory factors (IL-1β, TNF-α, IL-6).

[0026] (III) Effect of miR-491-5p knockdown on hPAEC cell proliferation

[0027] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. Samples were collected 48 hours later for detection, cell cloning, and EdU was used to detect the proliferation activity of hPAEC cells.

[0028] Conclusion: Knockdown of miR-491-5p promotes the proliferation of hPAEC cells.

[0029] (IV) Effect of miR-491-5p knockdown on hPAEC cell migration

[0030] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. Samples were collected 48 hours later for testing, and the migration activity of hPAEC cells was detected by scratch and Transwell-Migration assays.

[0031] Conclusion: Knockdown of miR-491-5p promotes the migration of hPAEC cells.

[0032] (V) Effect of miR-491-5p knockdown on hPAEC apoptosis

[0033] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. Samples were collected 48 hours later and the apoptotic activity of hPAEC cells was detected by TUNEL assay.

[0034] Conclusion: Knockdown of miR-491-5p inhibits hPAEC apoptosis.

[0035] Fourthly, investigate the effect of high expression of miR-491-5p on inflammatory factor levels and pulmonary artery endothelial cells

[0036] (I) Effect of high expression of miR-491-5p on the expression of pro-inflammatory genes

[0037] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. M1 induction was performed 48 h later, and the expression of pro-inflammatory genes (IL-1β, TNF-α, IL-6, CXCL10, CRP) in hPAEC cells was detected by RT-qPCR.

[0038] Conclusion: High expression of miR-491-5p promotes the levels of inflammatory factors.

[0039] (II) Effect of high expression of miR-491-5p on the secretion of inflammatory factors

[0040] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. After 48 hours, the supernatant of hPAEC cells was collected and the secretion levels of inflammatory factors IL-1β, TNF-α, and IL-6 were detected by ELISA.

[0041] Conclusion: High expression of miR-491-5p promotes the secretion of inflammatory factors.

[0042] (III) Effect of high expression of miR-491-5p on hPAEC cell proliferation

[0043] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. Cell cloning and EdU were used to detect the proliferation activity of hPAEC cells.

[0044] Conclusion: Overexpression of miR-491-5p inhibits the proliferation of hPAEC cells.

[0045] (IV) Effect of high expression of miR-491-5p on hPAEC cell migration

[0046] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. The migration activity of hPAEC cells was detected by scratch and Transwell-Migration.

[0047] Conclusion: Overexpression of miR-491-5p inhibits the migration of hPAEC cells.

[0048] (V) Effect of high expression of miR-491-5p on apoptosis of hPAEC

[0049] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. The apoptotic activity of hPAEC cells was detected by TUNEL assay.

[0050] Conclusion: High expression of miR-491-5p promotes hPAEC apoptosis.

[0051] Compared with the prior art, the beneficial effects of this application are:

[0052] This application discovered for the first time that miR-491-5p is highly expressed in the peripheral blood of sepsis patients and cecal ligation and puncture model mice; inhibiting miR-491-5p expression can inhibit inflammatory gene expression in hPAEC cells, inhibit the secretion of inflammatory factors, promote hPAEC cell proliferation, promote hPAEC cell migration, and inhibit hPAEC apoptosis; high expression of miR-491-5p can promote the level of inflammatory factors, promote the secretion of inflammatory factors, inhibit hPAEC cell proliferation, inhibit hPAEC cell migration, and promote hPAEC apoptosis.

[0053] In summary, inhibiting the expression of miR-491-5p provides a new possible treatment for sepsis combined with acute lung injury; high expression of miR-491-5p can provide a new possible method for constructing a sepsis combined with acute lung injury model. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 , expression of miR-491-5p in peripheral blood of patients with sepsis;

[0055] Figure 2 , miR-491-5p expression in peripheral blood, lung tissue, and kidney tissue of CLP mice, among which,

[0056] A is the expression of miR-491-5p in the peripheral blood of CLP mice.

[0057] B is the expression of miR-491-5p in lung tissue of CLP mice,

[0058] C is the expression of miR-491-5p in the kidney tissue of CLP mice;

[0059] Figure 3 , the expression levels of pro-inflammatory genes in hPAEC cells after miR-491-5p knockdown, among which,

[0060] A is the expression level of IL-1β,

[0061] B is the expression level of TNF-α,

[0062] C is the expression level of IL-6,

[0063] D is the expression level of CXCL10,

[0064] E is the expression level of CRP;

[0065] Figure 4 , the secretion level of inflammatory factors in hPAEC cells after miR-491-5p knockdown, where A is the secretion level of IL-1β,

[0066] B is the secretion level of TNF-α,

[0067] C is the secretion level of IL-6;

[0068] Figure 5 , detection of the proliferation ability of hPAEC cells after miR-491-5p knockdown, among which,

[0069] A is a cell clone,

[0070] B is the EdU experiment;

[0071] Figure 6 , the migration ability of hPAEC cells was detected after miR-491-5p knockdown, among which,

[0072] A is the scratch test,

[0073] B is Transwell-Migration cell migration assay;

[0074] Figure 7 , TUNEL assay to detect the apoptotic activity of hPAEC cells after miR-491-5p knockdown; Figure 8 , the expression levels of pro-inflammatory genes after high expression of miR-491-5p, where A is the expression level of IL-1β,

[0075] B is the expression level of TNF-α,

[0076] C is the expression level of IL-6,

[0077] D is the expression level of CXCL10,

[0078] E is the expression level of CRP;

[0079] Fig. 9 , the secretion level of inflammatory factors after high expression of miR-491-5p, among which,

[0080] A is the secretion level of IL-1β,

[0081] B is the secretion level of TNF-α,

[0082] C is the secretion level of IL-6,

[0083] Fig.10 , detection of proliferation ability of hPAEC cells with high expression of miR-491-5p, among which,

[0084] A is a cell clone,

[0085] B is the EdU experiment;

[0086] Fig.11 , the migration ability of hPAEC cells with high expression of miR-491-5p, where A is the scratch test,

[0087] B is Transwell-Migration cell migration assay;

[0088] Fig.12 , TUNEL assay to detect the apoptotic activity of hPAEC cells with high expression of miR-491-5p;

[0089] in Figure 1-Figure 12 middle,

[0090] *, **, *** and **** represent P<0.05, P<0.01, P<0.001 and P<0.0001, respectively, indicating that the difference between the two groups is statistically significant. The more *s there are, the greater the statistical difference. DETAILED DESCRIPTION

[0091] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0092] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0093] All materials, reagents, etc. in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0094] Experimental Materials

[0095]

[0096]

[0097] Experimental equipment

[0098]

[0099] Example 1. Expression of miR-491-5p in peripheral blood of patients with sepsis

[0100] 1. Methods (blood RNA extraction, RT-qPCR)

[0101] Peripheral blood samples were collected from normal subjects (n=30) and sepsis patients (n=30), and total RNA was extracted using Trizol Reagent reagent. RNA quality was tested and quantified. 2000 ng RNA was reverse transcribed into cDNA, and cDNA, qPCR primers, 2x SuperReal PreMix Plus reagent, and RNase-Free ddH2O were added to a 96-well plate. The 96-well plate was placed in a qPCR instrument, parameters were set, and the instrument was started. RT-qPCR was used to detect the expression level of miR-491-5p.

[0102] 2. Results

[0103] like Figure 1 As shown, the expression of miR-491-5p in peripheral blood samples of sepsis patients and normal subjects was significantly different (P < 0.0001), and miR-491-5p was highly expressed in sepsis patients.

[0104] Example 2. Expression of miR-491-5p in the peripheral blood of CLP mice

[0105] 1. Methods (total RNA extraction from peripheral blood samples, lung tissue, and kidney tissue, RT-qPCR)

[0106] Construction of mouse cecal ligation and puncture (CLP) model (n=10) Methods: After anesthesia, C57BL / 6J black mice were anesthetized, the abdominal skin was prepared, disinfected, and covered with a towel. An incision of about 1.5 cm was made in the lower abdomen along the midline of the abdomen. The root of the cecum was ligated with No. 1 thread, and the cecum was punctured twice with an 18-gauge injection needle. After squeezing out the contents of the cecum, the peritoneum and skin were sutured to obtain the cecal ligation and puncture model mice.

[0107] Total RNA was extracted from peripheral blood samples, lung tissues, and kidney tissues of cecal ligation and puncture model mice and normal mice, and the expression level of miR-491-5p was detected by RT-qPCR. The RT-qPCR detection method is shown in Example 1.

[0108] 2. Results

[0109] like Figure 2 As shown, there were significant differences in expression in peripheral blood samples, lung tissues, and kidney tissues between cecal ligation and puncture model mice and normal mice, with P values ​​all less than 0.0001. miR-491-5p was highly expressed in peripheral blood samples, lung tissues, and kidney tissues of cecal ligation and puncture model mice.

[0110] Example 3 Effects of miR-491-5p knockdown on inflammatory factor levels and pulmonary artery endothelial cells (hPAEC cells)

[0111] 1. Effect of miR-491-5p knockdown on pro-inflammatory gene expression

[0112] 1. Methods

[0113] 1. Knockdown of miR-491-5p

[0114] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. After the hPAEC cells were full grown, the cell viability was counted and tested. After the cell viability reached 90%, the cells were plated for the experiment. The next day, the miR-491-5p-inhibitor was mixed with the jetPRIME transfection reagent Transfected Reagent (Polyplus transfection reagent), and the mixed RNA mixture was added to the culture dish. After 6 hours, it was replaced with ordinary complete culture medium to obtain hPAEC cells with knockdown of miR-491-5p. M1 induction was performed after 48 hours.

[0115] 2. M1 induction

[0116] hPAEC cells transfected with control irrelevant sequence NC inhibitor and knocked down miR-491-5p (miR-491-5p-inhibitor cells) were prepared and uniformly induced with PMA 200 ng / ml for 72 h. After induction to M0, medium containing IFN-γ and LPS were added to induce for 48 h to M1, and the hPAEC cells were collected.

[0117] 3. Detection of pro-inflammatory genes (IL-1β, TNF-α, IL-6, CXCL10, CRP) in hPAEC cells

[0118] hPAEC cells were collected, total RNA was extracted, and RT-qPCR was used to detect the transcriptional expression levels of pro-inflammatory genes IL-1β, TNF-α, IL-6, CXCL-10, and CRP in hPAEC cells. The RT-qPCR detection method is shown in Example 1.

[0119] (II) Results

[0120] like Figure 3As shown, in hPAEC cells, compared with the normal group (NC inhibitor M0), the expression of hPAEC cell proinflammatory genes (IL-1β, TNF-α, IL-6, CXCL10, CRP) in the NC inhibitor M1 proinflammatory group (M1 represents the proinflammatory effect of Lps-stimulated cells) was significantly increased; miR-491-5p inhibitor M1 (miR-491-5p inhibitor represents knockdown of miR-491-5p) inhibited the expression of hPAEC cell proinflammatory genes, or restored the expression of hPAEC cell proinflammatory genes to a certain level.

[0121] 3. Conclusion

[0122] miR-491-5p knockdown inhibits pro-inflammatory gene expression.

[0123] 2. Effect of miR-491-5p knockdown on the secretion of inflammatory factors

[0124] 1. Methods

[0125] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. After the hPAEC cells were full grown, the cell viability was detected and the cells were plated for the experiment when the cell viability reached 90%. The miR-491-5p-inhibitor was mixed with the transfection reagent the next day, and the mixed RNA mixture was added to the culture dish. After 6 hours, it was replaced with ordinary complete culture medium. After 48 hours, the hPAEC cell supernatant was collected and the secretion levels of inflammatory factors IL-1β, TNF-α, and IL-6 were detected by ELISA.

[0126] (II) Results

[0127] like Figure 4 As shown, in hPAEC cells, compared with the normal group (NC inhibitor M0), the secretion of hPAEC cell inflammatory factors (IL-1β, TNF-α, IL-6) in the NC inhibitor M1 pro-inflammatory group (M1 represents the pro-inflammatory effect of Lps-stimulated cells) was significantly increased; miR-491-5p inhibitor M1 (miR-491-5pinhibitor represents knockdown of miR-491-5p) inhibited the secretion of hPAEC cells inflammatory factors, or restored the secretion of hPAEC cells inflammatory factors to a certain level.

[0128] 3. Conclusion

[0129] Knockdown of miR-491-5p inhibited the secretion of inflammatory factors.

[0130] Effect of miR-491-5p knockdown on hPAEC cell proliferation

[0131] 1. Methods

[0132] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. After the hPAEC cells were full grown, the cell viability was detected and the cells were plated for the experiment when the cell viability reached 90%. The miR-491-5p-inhibitor was mixed with the transfection reagent the next day, and the mixed RNA mixture was added to the culture dish. After 6 hours, it was replaced with ordinary complete culture medium, and samples were collected for detection after 48 hours.

[0133] 1. Cell cloning

[0134] The gelatin was coated in a 6-well plate and cells were plated after coating for 30 min at room temperature. The cells were resuspended and counted. The cell number was 2×10 3 / well was added to a 6-well plate, mixed thoroughly, and allowed to stand for 10-20 minutes. After the cells adhered to the wall, the culture dish was placed in a 37°C, 5% CO2 incubator for 14 days. After 14 days, the culture dish was removed, the cells were washed with PBS, 1 ml of fixative was added for 15 minutes, and then 0.1% crystal violet staining solution was added for 15 minutes. The residual crystal violet was washed with tap water, the plate was dried, photographed, and the number of clones was counted.

[0135] 2. EdU experiment

[0136] Follow BeyotCLick TM EdU detection was performed using the EdU-594 cell proliferation detection kit.

[0137] (II) Results

[0138] like Figure 5 As shown, in hPAEC cells, compared with the normal group (NC inhibitor M0), the proliferation ability of hPAEC cells in the NC inhibitor M1 pro-inflammatory group (M1 represents the pro-inflammatory effect of Lps-stimulated cells) was significantly weakened; miR-491-5pinhibitor M1 (miR-491-5p inhibitor represents knockdown of miR-491-5p) promoted the proliferation activity of hPAEC cells, or restored the proliferation function of hPAEC cells to a certain level.

[0139] 3. Conclusion

[0140] Knockdown of miR-491-5p promoted the proliferation of hPAEC cells.

[0141] Effect of miR-491-5p knockdown on hPAEC cell migration

[0142] 1. Methods

[0143] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. After the hPAEC cells were full grown, the cell viability was detected and the cells were plated for the experiment when the cell viability reached 90%. The miR-491-5p-inhibitor was mixed with the transfection reagent the next day, and the mixed RNA mixture was added to the culture dish. After 6 hours, it was replaced with ordinary complete culture medium, and samples were collected for detection after 48 hours.

[0144] 1. Scratches

[0145] The cells were plated into 6-well plates. The next day, old culture medium was removed with a 1 ml pipette. A straight line was drawn from top to bottom in the middle of the well with a 200 ul yellow pipette tip. 2 ml culture medium was slowly added along the well plate wall. Immediately, photographs were taken under a microscope (4X objective lens) and recorded as migration at 0 h. After 24 h, the medium replacement step was repeated and photographs were taken under a microscope (4X objective lens) and recorded as migration at 24 h.

[0146] 2. Transwell-Migration

[0147] Add 200ul serum-free culture medium to the Transwell chamber and place it in the incubator for more than 30 minutes to infiltrate the chamber. Take out the infiltrated chamber from the incubator, add 500ul / well serum to the lower chamber, add 200ul of cells with adjusted density to the chamber, and place the 24-well plate in the incubator for 24 hours. Set the experimental wells and collect samples after 24 hours of migration.

[0148] (II) Results

[0149] like Figure 6 As shown, in hPAEC cells, compared with the normal group (NC inhibitor M0), the migration ability of hPAEC cells in the NC inhibitor M1 pro-inflammatory group (M1 represents the pro-inflammatory effect of Lps-stimulated cells) was significantly weakened; miR-491-5pinhibitor M1 (miR-491-5p inhibitor represents knockdown of miR-491-5p) promoted the migration activity of hPAEC cells, or restored the migration function of hPAEC cells to a certain level.

[0150] 3. Conclusion

[0151] Knockdown of miR-491-5p promotes hPAEC cell migration.

[0152] 5. Effect of miR-491-5p knockdown on hPAEC apoptosis

[0153] (I) Method (TUNEL)

[0154] The expression of miR-491-5p was inhibited by transfecting hPAEC cells with miR-491-5p-inhibitor. After the hPAEC cells were full grown, the cell viability was detected and the cells were plated for the experiment when the cell viability reached 90%. The miR-491-5p-inhibitor was mixed with the transfection reagent the next day, and the mixed RNA mixture was added to the culture dish. After 6 hours, it was replaced with ordinary complete culture medium, and samples were collected for detection after 48 hours.

[0155] TUNEL detection was performed according to the Bio-Tech One-Step TUNEL Cell Apoptosis Detection Kit.

[0156] (II) Results

[0157] like Figure 7 As shown, in hPAEC cells, compared with the normal group (NC inhibitor M0), the apoptosis ability of hPAEC cells in the NC inhibitor M1 pro-inflammatory group (M1 represents the pro-inflammatory effect of Lps-stimulated cells) was significantly increased; miR-491-5pinhibitor M1 (miR-491-5p inhibitor represents knockdown of miR-491-5p) inhibited the apoptotic activity of hPAEC cells, or restored the apoptotic function of hPAEC cells to a certain level.

[0158] 3. Conclusion

[0159] miR-491-5p knockdown inhibits hPAEC apoptosis.

[0160] Example 4. Effect of high expression of miR-491-5p on inflammatory factor levels and pulmonary artery endothelial cells (hPAEC cells)

[0161] 1. Effect of high expression of miR-491-5p on the expression of pro-inflammatory genes

[0162] (I) Methods (miR-491-5p overexpression, induction of M1, RT-qPCR)

[0163] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. After the hPAEC cells were confluent, the cell viability was detected. After the cell viability reached 90%, the cells were plated for the experiment. The next day, miR-491-5p-mimic was mixed with the transfection reagent, and the mixed RNA mixture was added to the culture dish. After 6 hours, it was replaced with ordinary complete culture medium, and M1 induction was performed after 48 hours.

[0164] The M1 induction experimental method is shown in Example 3, and the RT-qPCR detection method is shown in Example 1.

[0165] (II) Results

[0166] like Figure 8 As shown, in hPAEC cells, compared with the normal group (NC mimic M0), the expression of hPAEC cell proinflammatory genes (IL-1β, TNF-α, IL-6, CXCL10, CRP) was significantly increased in the NC mimic M1 proinflammatory group (M1 represents the proinflammatory effect of Lps-stimulated cells); miR-491-5p mimic M1 (miR-491-5p mimic represents high expression of miR-491-5p) further promoted the expression of hPAEC cell proinflammatory genes.

[0167] 3. Conclusion

[0168] High expression of miR-491-5p promotes the levels of inflammatory factors.

[0169] 2. Effect of high expression of miR-491-5p on the secretion of inflammatory factors

[0170] 1. Methods

[0171] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. After the hPAEC cells were full grown, the cell viability was detected and the cells were plated for the experiment when the cell viability reached 90%. The next day, miR-491-5p-mimic was mixed with the transfection reagent and the mixed RNA mixture was added to the culture dish. After 6 hours, it was replaced with ordinary complete culture medium. After 48 hours, the hPAEC cell supernatant was collected and the secretion levels of inflammatory factors IL-1β, TNF-α, and IL-6 were detected by ELISA.

[0172] (II) Results

[0173] like Fig. 9 As shown, in hPAEC cells, compared with the normal group (NC mimic M0), the secretion of hPAEC cell inflammatory factors (IL-1β, TNF-α, IL-6) in the NC mimic M1 pro-inflammatory group (M1 represents the pro-inflammatory effect of Lps-stimulated cells) was significantly increased; miR-491-5p mimic M1 (miR-491-5p mimic represents high expression of miR-491-5p) further promoted the secretion of hPAEC cells inflammatory factors.

[0174] 3. Conclusion

[0175] High expression of miR-491-5p promotes the secretion of inflammatory factors.

[0176] 3. Effect of high expression of miR-491-5p on the proliferation of hPAEC cells

[0177] (I) Methods (cell cloning, EdU)

[0178] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. The method is shown in "I. Effect of high expression of miR-491-5p on the expression of pro-inflammatory genes" in Example 4, and the cell cloning and EdU method are shown in Example 3.

[0179] (II) Results

[0180] like Fig.10 As shown, in hPAEC cells, compared with the normal group (NC mimic M0), the proliferation ability of hPAEC cells in the NC mimic M1 pro-inflammatory group (M1 represents the pro-inflammatory effect of Lps-stimulated cells) was significantly weakened; miR-491-5p mimic M1 (miR-491-5p mimic represents high expression of miR-491-5p) further inhibited the proliferation activity of hPAEC cells.

[0181] 3. Conclusion

[0182] High expression of miR-491-5p inhibits the proliferation of hPAEC cells.

[0183] 4. Effect of high expression of miR-491-5p on the migration of hPAEC cells

[0184] (I) Method (scratch, transwell-migration)

[0185] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. The method is shown in "I. Effect of high expression of miR-491-5p on the expression of pro-inflammatory genes" in Example 4, and the scratch and Transwell-Migration methods are shown in Example 3.

[0186] (II) Results

[0187] like Fig.11 As shown, in hPAEC cells, compared with the normal group (NC mimic M0), the migration ability of hPAEC cells in the NC mimic M1 pro-inflammatory group (M1 represents the pro-inflammatory effect of Lps-stimulated cells) was significantly weakened; miR-491-5p mimic M1 (miR-491-5p mimic represents high expression of miR-491-5p) further inhibited the migration activity of hPAEC cells.

[0188] 3. Conclusion

[0189] Overexpression of miR-491-5p inhibited the migration of hPAEC cells.

[0190] 5. Effect of high expression of miR-491-5p on apoptosis of hPAEC

[0191] (I) Method (TUNEL)

[0192] The expression of miR-491-5p was increased by transfecting hPAEC cells with miR-491-5p-mimic. The method is shown in "I. Effect of high expression of miR-491-5p on the expression of pro-inflammatory genes" in Example 4, and the TUNEL experimental method is shown in Example 3.

[0193] (II) Results

[0194] like Fig.12 As shown, in hPAEC cells, compared with the normal group (NC mimic M0), the apoptosis ability of hPAEC cells in the NC mimic M1 pro-inflammatory group (M1 represents the pro-inflammatory effect of Lps-stimulated cells) was significantly increased; miR-491-5p mimic M1 (miR-491-5p mimic represents high expression of miR-491-5p) further promoted the apoptotic activity of hPAEC cells.

[0195] 3. Conclusion

[0196] High expression of miR-491-5p promotes hPAEC apoptosis.

[0197] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. Use of an agent for inhibiting the expression of miR-491-5p in hPAEC cells in the preparation of a drug for treating sepsis combined with acute lung injury, characterized in that: The reagent inhibits the expression of miR-491-5p by transfecting hPAEC cells with miR-491-5p-inhibitor.

2. The use according to claim 1, characterized in that The reagent promotes the proliferation activity of hPAEC cells, promotes the migration activity of hPAEC cells, and inhibits the apoptosis of hPAEC cells.

3. Use of an agent for inhibiting the expression of miR-491-5p in hPAEC cells in the preparation of a drug for treating sepsis, characterized in that: The reagent inhibits the expression of miR-491-5p by transfecting hPAEC cells with miR-491-5p-inhibitor.

4. The use according to claim 2, characterized in that The reagent inhibits the expression of pro-inflammatory genes IL-1β, TNF-α, IL-6, CXCL10, and CRP in hPAEC cells; and inhibits the secretion of inflammatory factors IL-1β, TNF-α, and IL-6 in hPAEC cells.

5. Use of an agent that increases the expression of miR-491-5p in hPAEC cells in promoting the progression of sepsis combined with acute lung injury, characterized in that: The reagent increases the expression of miR-491-5p by transfecting hPAEC cells with miR-491-5p-mimic, and the application is for non-disease diagnosis and treatment purposes.

6. The reagent according to claim 5, characterized in that The reagent inhibits the proliferation activity of hPAEC cells, inhibits the migration activity of hPAEC cells, and promotes the apoptosis of hPAEC cells.

7. Use of an agent that increases miR-491-5p expression in hPAEC cells in promoting the progression of sepsis, characterized in that: The reagent increases the expression of miR-491-5p by transfecting hPAEC cells with miR-491-5p-mimic, and the application is for non-disease diagnosis and treatment purposes.

8. The reagent according to claim 7, characterized in that The reagent promotes the expression of pro-inflammatory genes IL-1β, TNF-α, IL-6, CXCL10, and CRP in hPAEC cells; and promotes the secretion of inflammatory factors IL-1β, TNF-α, and IL-6 in hPAEC cells.

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