Circulating Blood miR-155 and miR-21 Promote the Development of Acute Pancreatitis and Can Be Used to Assess the Risk Stratification of Pancreatitis

To explore the role of circulating blood miR-155 and miR-21 in promoting acute pancreatitis (AP) and evaluating the risk stratification of pancreatitis. In this experiment, 70 patients with AP treated in our hospital from October 2019 to December 2020 were included in the research group (RG), and the blood of 52 healthy cases was collected and they were included in the control group (CG). The expression of miR-155 and miR-21 in circulating blood was observed in the two groups. The diagnostic efficacy of miR-155 and miR-21 in AP was observed. The risk factors of patients with AP were observed. The expression of serum gastrointestinal hormones was observed in the two groups. The GAS and VIP in RG were higher than those in CG, while MTL and CCK were lower than those in CG. Moreover, the detection level of mild, moderate, severe, and critical patients was also significantly different (P < 0.05). The expression of miR-155 and miR-21 in circulating blood of RG was significantly lower than that of CG (P < 0.05), and the area under the miR-155 curve was 0.775 and the area under the miR-21 curve was 0.832. Alcoholism, GAS, VIP, MTL, CCK, miR-155, and miR-21 were the risk factors of patients. miR-155 and miR-21 show low expression in the serum of patients. The lower the expression, the more serious the disease. They are closely related to the development of AP. miR-155 and miR-21 have good diagnostic efficacy by ROC analysis, and they are expected to become effective indicators for the diagnosis and treatment of AP in the future.


Introduction
Pancreatitis is a major cause of gastrointestinal disease-related diseases, which is related to high morbidity, mortality, and social and economic burden [1]. e incidence of AP is different all over the world [2]. According to WHO definition [3], the high-incidence areas (i.e., areas with an annual incidence exceeding 34 cases per 100,000 people) are North America and Western Pacific. As a whole, Europe is a low-incidence area (there are 29 cases per 100,000 ordinary people every year). It has been revealed that the incidence of AP varies throughout the European continent, and Northern Europe and Eastern Europe are the most affected [4]. Nevertheless, due to the lack of excellent research from Eastern and Southern Europe, it is difficult to compare the morbidity of AP in Europe in a powerful way [5]. In view of this situation, we urgently need to find a new potential target for treatment and diagnosis and further clarify the development of pancreatitis to improve the current situation.
With the deepening of research, scholars at home and abroad began to focus on gene research in recent years. miRNA (microRNA) is a kind of small noncoding RNA, which can be applied as endogenous RNA interference to adjust the targeted genes and affect the regulation of various physiology and pathologic functions [6]. Bioinformatics data reveal that a single miRNA can bind to hundreds of target mRNA, thus playing a crucial role in various biological processes. At present, it has been proved to be closely related to the development and progression of various human tumor diseases [7,8]. miRNA is a protein regulator, which plays an important role in various cell functions [9]. miR-155 has a series of known biological functions, including inducing the activation of Toll-like receptors (TLR) and regulating TLR signaling transduction in monocytes/macrophages, thus promoting inflammatory cell reactions and triggering systemic inflammatory reactions [10,11]. Research studies have revealed that [12] miR-21 is also closely bound up with the development and progression of pancreatitis. erefore, we will verify our conjecture through experimental analysis, so as to provide references and guidance for clinical diacrisis and therapy of pancreatitis in the future. Our study aims to explore the role of circulating blood miR-155 and miR-21 in promoting acute pancreatitis (AP) and evaluating the risk stratification of pancreatitis.

Baseline Data.
From October 2019 to December 2020, 70 cases with AP admitted to our hospital were included in the RG and the blood of 52 healthy cases was collected and they were included in the CG in this experiment. In RG, there were 41 men and 29 women with a mean age of 46.6 ± 4.3 years. Disease degree: mild symptoms, there were 17 mild cases, 20 moderate cases, 18 severe cases, and 15 critical cases. In CG, there were 28 men and 24 women with a mean age of 46.2 ± 4.7 years. e collection of clinical data and this test were conducted and ratified by the Medical Ethics Committee of our hospital.

Inclusive and Exclusive Criteria.
Inclusive criteria included the following: patients who met the comprehensive diagnosis standard for AP issued by the Chinese Association of Integrated Traditional Chinese and Western Medicine [13]; patients who did not take drugs that affected the test results; patients who cooperated with medical workers to carry out research; patients whose age was 30∼60 years and they had full case materials; patients who were disposed to cooperate with the working schedule of medical workers in our hospital; patients or their immediate dependents who affixed the informed consent form.
Exclusive criteria were as follows: death during therapy, damage to important organs, complicated with other cardiocerebrovascular diseases, deformity in body, pregnancy, complicated with other autoimmunity diseases, complicated with other chronic illnesses, patients who moved to other hospitals, contraindication of operation, mental disorders, language disturbance, and diseases affecting the findings of this research.

Collection of Blood Samples.
In the early morning, 5 ml of fasting peripheral vein blood was collected from the two groups. Among them, 2 mL of blood samples was stored at 4°C for 30 minutes and centrifuged for 10 minutes (3000 rpm/m). en, the supernatant fluid was obtained and conserved in a refrigerator at −80°C. In addition, 3 mL of blood samples was tested for miR-155 and miR-21, all of which were processed by the qRT-PCR amplification method.

qRT-PCR Detection and ELISA Detection.
e blood samples stored at −80°C were taken out, and the total RNA of tissues was extracted by the TRIzol kit. e purity, concentration, and integrality of the obtained general RNA were tested via ultraviolet spectrophotometry and agarose gel electrophoresis. TransScript Green miRNA Two-Step qRT-PCR SuperMix (AQ202-01, Beijing TransGen Biotech Company, China) was applied for a reverse transcript of the extracted general RNA. e test was conducted according to the kit instructions, and the cDNA was collected for the PCR amplified experiment. e qPCR amplified system was as follows: cDNA (1 μL), upstream and downstream primers (each 0.4 μL), 2 × TransTaq ® Tip Green qPCR SuperMix (10 μL), and Passive Reference Dye (50X) (0.4 μL). In the end, ddH 2 O was added to complete to 20 μL. e qPCRamplified procedures were initial denaturation at 94°C for 30 s, degeneration at 94°C for 5 s, and annealing and extending at 60°C for 30 s, with 40 cycles. ree duplicate holes were established for each specimen, and the test was conducted 3 times in total. In this research, U6 was applied as an internal parameter and 2 −△ct was applied to analyze this data. GAS, VIP, MTL, and CCK were obtained in strict accordance with ELISA kit specifications.

Outcome Measures.
e main outcome measures were as follows: the miR-155 and miR-21 in circulating blood were observed in the two groups. e diagnostic efficacy of miR-155 and miR-21 in AP was observed. e risk factors of patients with AP were observed. e secondary outcome measures were as follows: the expression of serum gastrointestinal hormones was observed in the two groups.

Statistical Analysis.
In this research, SPSS20.0 was used to statistically analyze the data. GraphPad 7 was applied to plot the requisite pictures. e K-S test was applied to analyze the distribution of dose data, in which normal distribution data were represented via mean number ± standard error (mean ± SD). e independent-samples t-test was applied for comparison between groups. e paired t-test was applied for comparison within groups.
e enumeration data were represented as a percentage. Chi-square tests were applied, denoted by χ 2 . ROC was applied to plot the diagnosis efficacy of miR-155 and miR-21 in AP. e single-factor and multifactor logistic regression were applied to analyze the risk factors of patients. ere were statistical differences with P < 0.05.

Clinical Materials.
ere was no obvious difference in clinical data such as age, gender, BMI, smoking history, drinking history, place of residence, exercise habit, and marital status between RG and CG, which proved comparability (P > 0.05) ( Table 2).

Observing the Expression of Serum Gastrointestinal
Hormones in the Two Groups.
e GAS and VIP in RG were higher than those in CG, while MTL and CCK were lower than those in CG. Moreover, the detection level of mild, moderate, severe, and critical patients was also obviously different (P < 0.05) (Table 3, Figure 1).

Observing the miR-155 and miR-21 in Circulating Blood between the Two Groups.
rough qRT-PCR detection, the miR-155 and miR-21 in circulating blood of RG were 0.53 ± 0.46 and 0.81 ± 0.21, while those of CG were 2.86 ± 0.46 and 2.33 ± 0.57. e RG was obviously lower than the CG (P < 0.05) (Figure 2).

Observing the Diagnostic Efficacy of miR-155 and miR-21 in AP.
According to the expressions of miR-155 and miR-21, ROC was plotted. e findings revealed that the area under the miR-155 curve was 0.775 (P < 0.05) and the area under the miR-21 curve was 0.832 (P < 0.05) (Table 4, Figure 3).

Univariate Logistic Regression Analysis.
According to the degree of illness, the patients were divided into 37 cases with mild or moderate disease and 33 cases with severe and critical illness. Univariate analysis was carried out according to the clinical data in the mild and moderate degree group and severe and critical group. e results revealed that there were no differences in age and gender (P > 0.05), but there were differences in alcoholism, GAS, VIP, MTL, CCK, miR-155, and miR-21 (P < 0.05) ( Table 5).

Discussion
AP is an inflammation-induced disease of the pancreas, and the most general cause is gallstones or excessive use of alcohol. AP can range from slight self-limiting diseases requiring only supportive measures to serious diseases with life-threatening complications [14]. Severe illness accounts for about 20-30% of patients, which is a lifethreatening disease with a mortality rate of about 15%. e most commonly used taxonomy system of AP is the Atlanta classification and definition revised in 2012 based on international consensus. It has been identified into two stages (early and late) by this classification. e order of severity is divided into mild, moderate, severe, and critical. Mild patients (interstitial edematous pancreatitis) have no organ failure and local or systemic complications, and the disease usually goes away within the first week. If there is transient (less than 48 hours) organ failure, local complications, or deterioration of the   [15]. Effective biological indicators for diagnosis and treatment of AP are a major research focus in clinics at present.
In this study, miR-155 and miR-21 in patients' serum were tested and the results showed that the miR-155 and miR-21 of patients in RG were lower than those in CG, which revealed that miR-155 and miR-21 were closely related to the development and progression of AP. miR-155, a multifunctional miRNA with  inflammatory-related functions, is adjusted by different inflammation media. Bacterial lipopolysaccharide (LPS) and inflammation media, including IFN-β and TNF-α, can induce miR-155 in human monocyte cell strain. Especially, because it participates in the molecular changes of key targets and signal pathways, the imbalance of miR-155 has been closely related to Helicobacter pylori-related gastropathy, inflammatory intestinal disease, and colorectal carcinoma [16][17][18]. Studies [12,19,20] have shown that AP induced by protein in wild-type (WT) mice receiving miR-21 gene knockout (KO) bone marrow is semblable to WT mice transplanted with WT bone marrow, which excludes the possibility of inflammatory invasion. Macrophages    or other leukocytes play an important part in the phenotype of less-severe AP in miR-21 KO mice. erefore, we further mapped ROC. e findings revealed that when the cutoff value was 0.780, the sensitivity and specificity of miR-155 were 67.31% and 78.57%. When the cutoff value was 0.931, the sensitivity and specificity of miR-21 were 86.42% and 71.43%, respectively. ey have high diagnostic efficiency, suggesting that miR-155 and miR-21 are expected to be effective targets in diagnosing AP in the future. e expression of serum gastrointestinal hormones was observed in both groups. e findings revealed that GAS and VIP in RG were higher than those in CG, while MTL and CCK were lower than those in CG. ere were also significant differences in the detection level of mild, moderate, severe, and critical patients, which indicated that the higher the expression of GAS and VIP, the more serious the patient's condition, while MTL and CCK were opposite. Studies have shown that [21] the age-standardized mortality rate of pancreatitis in China has not changed since 1990. For pancreatitis, alcoholism, obesity, diabetes, and hypertriglyceridemia are some of the risk factors [22], and East-Asian populations are appropriately aware of the risk factors for AP such as regulated metabolism and lifestyle, so it may be different from the western population because of the different relationships between obesity and insulin resistance [23]. At last, we conducted logistic regression analysis and concluded that alcoholism, GAS, VIP, MTL, CCK, miR-155, and miR-21 were the risk factors of patients with AP.

Conclusion
We have preliminarily revealed the clinical significance of miR-155 and miR-21 through the above research, but there are still some limitations in our study. Firstly, we did not  Yes � 1, no � 0 GAS (μmol/L) Data belonged to continuous variables and were analyzed by original data VIP (μmol/L) Data belonged to continuous variables and were analyzed by original data MTL (pg/ml) Data belonged to continuous variables and were analyzed by original data CCK (pg/ml) Data belonged to continuous variables and were analyzed by original data miR-155 Data belonged to continuous variables and were analyzed by original data miR-21 Data belonged to continuous variables and were analyzed by original data conduct basic cell experiments in this study. Secondly, we did not conduct targeted drug therapy, so we hope to supplement our research results by conducting basic cell experiments and targeted drug therapy in future studies. In summary, miR-155 and miR-21 show low expression in AP, suggesting that miR-155 and miR-21 are involved in the occurrence and development of AP. erefore, they are expected to be effective indicators to diagnose and treat AP and to judge the prognosis of patients in the future.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.