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Long Non-Coding RNA (lncRNA) SNHG5 Participates in Vertical Sleeve Gastrectomy for Type II Diabetes Mellitus by Regulating TGR5

Background Due to its remarkable effect in controlling glycometabolism, relatively simple operation, and low risk of complications, sleeve gastrectomy (SG) has become the preferred surgical treatment for type II diabetes mellitus. Increased blood glucose in the body can cause damage to functional cells. Material/Methods Long non-coding RNA SNHG5 expression and TGR5 in serum were analyzed by real-time PCR. A diabetic cell model was established by culturing normal human intestinal epithelial cells NCM460 and DLD-1 with high-glucose and high-fat medium. CCK-8 assay, TUNEL assay, and flow cytometry were used to assess cell growth and apoptosis, respectively. The secretion of lactate dehydrogenase (LDH) was detected using the LDH Cytotoxicity Kit. lncRNA SNHG5 was downregulated by siRNA. The changes in expression of SNHG5, TGR5, Akt, p65, and Bcl-2 were analyzed by real-time PCR assay or Western blot. Results In 40 type II diabetes patients who underwent sleeve gastrectomy, the expression of SNHG5 decreased and the expression of TGR5 increased compared with that before the operation. After high-glucose and high-fat culture, cell growth was inhibited and cell apoptosis increased significantly. The expression of SNHG5 was increased and TGR5 was decreased with high-glucose and high-fat culture. However, high glucose and high fat showed an opposite trend for cell growth, apoptosis, and LDH release under inhibition of SNHG5. The expression levels of TGR5 and Akt, p65, and Bcl-2 were also returned to normal by SNHG5 inhibition. Conclusions By downregulating expression of the SNHG5 gene and then altering expression of the TGR5 gene, the damage to colorectal cells induced by high glucose was alleviated. This may be one of the mechanisms underlying the effect of sleeve gastric surgery in treatment of diabetes mellitus.


Background
Diabetes mellitus is a progressive systemic disease caused by uncontrolled high blood glucose levels, with many serious complications. The main pathogenesis of diabetes mellitus is that the body loses the ability to regulate blood glucose, which results in the increase of blood glucose and damages functional cells. With economic and social development, the improvement of living standards, and the longer life expectancy, the incidence of diabetes mellitus is increasing rapidly, and most cases are type II diabetes [1], which has become very serious health problem worldwide. Therefore, the aim of treatment for type II diabetes mellitus is to make blood glucose and other indicators reach target levels as soon as possible, eliminate the effect of glucose toxicity, restore and protect the function of insulin secretion, and delay and avoid the occurrence of complications [2]. Sleeve gastrectomy has achieved good results in the surgical treatment of type II diabetes for years. Sleeve gastrectomy consists of cutting out the great curvature of the stomach by laparoscopy, so that the stomach has a small gastric sac, which can reduce the volume of the stomach and thus reduce the secretion of hormones stimulated by hunger. Sleeve gastrectomy is suitable for patients who have had type II diabetes for less than 15 years, whose islet cells retain some insulin secretion ability, and with fasting serum C peptide level higher than 2/1 of the lower limit of normal value, BMI ³27.5 kg/m 2 , male waist ³90 cm, female waist ³85 cm, and for whom the operation is recommended as appropriate.
Long non-coding RNAs (lncRNAs) have transcripts of more than 200 nt. These transcripts have structural similarity to mRNAs, but their functions are different from those of RNA, such as cis-regulation ability and lack of open reading frames [3]. Gene expression in epigenetic regulation is regulated by lncRNAs, as well as transcriptional regulation and post-transcriptional regulation, and provide structural integrity for cells [4]. For example, lncRNA H19 is a prognostic factor in pancreatic neuroendocrine neoplasms (pNENs) and functions as an oncogene through the VGF-mediated PI3K/AKT/CREB pathway [5]. lncRNA SNHG14 is highly expressed in cervical tumor tissues and cells, and promotes the progression of cervical cancer [6]. Recent studies found that lncRNA has a regulatory function in the occurrence and development of diabetes mellitus. Overexpression of lncRNA TGU1 upregulates the expression of peroxisome proliferator-activated receptor g coactivator-1a (PGC-1a), reduces mitochondrial damage induced by high glucose levels, and alleviates the kidney damage caused by diabetes mellitus [7].
We investigated the effects of SNHG5 on high-glucose-induced colorectal cell damage and its expression in type II diabetic patients after sleeve gastric surgery. The effects of knockdown of SNHG5 were investigated by culturing normal human intestinal epithelial cells NCM460 and DLD-1 with high glucose and high fat, assessing cell growth and apoptosis using SNHG5 small interfering RNA (siRNA). We also explored tested its signal pathway in a diabetic cell model.

Material and Methods
Patients and cell lines Forty type II diabetes mellitus patients were included in this study. All these selected patients were diagnosed with type II diabetes and treated in our hospital from March 2016 to March 2018. The criteria for type II diabetes were: random plasma glucose ³11.1 mmol/l polyuria, fatigue, polydipsia, or weight loss, and 2-h post-load glucose ³11.1 mmol/l following 75 g oral glucose intake. Patient selection criteria were: diagnosed with type II diabetes for the first time and without complications, completed follow-up for at least 1 year, and underwent sleeve gastrectomy in our hospital after the diagnosis of type II diabetes mellitus. There were 24 males and 16 females, with a mean age of 33.5±5.5 years old (range 15-62 years). Without drug treatment after sleeve gastrectomy, HbA1c £6%, fasting blood glucose £7.0 mmol/L, and PG £10 mmol/L 2 h after meal. Blood samples were taken from all patients before sleeve gastrectomy, which was also performed 6 months and 12 months after the operation. This study was approved by the Ethics Committee of the First Affiliated Hospital of JiaMusi University. NCM460 (human normal intestinal epithelial) and DLD-1 (human colorectal adenocarcinoma epithelial cells) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured with Roswell Park Memorial Institute-1640 (RPMI-1640, Gibco, Grand Island, NY) culture medium containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) at 37°C with 5% CO 2 . RPMI-1640 medium containing 25 mmol/L glucose and 500 mmol/L of saturated free fatty acid palmitate [16: 0] was used to induce cell injury and create the cell model. Because there is no standard for creating a diabetic cell model in colorectal cells, we choose specific drugs and high-glucose treatment cells to detect cell viability, apoptotic rate, and LDH release as indicators of cell damage induced by high glucose. Lipofectamine 2000 (Life Technologies, CA, USA) was used to transfect SNHG5specific small interfering RNA (siRNA) and scrambled siRNA (GenePharma Company, Shanghai, China) into NCM460 following the manufacturer's instructions. NCM460 cells in the high-glucose and high-fat group were treated with 75 mmol/L glucose and 300 mmol/L palmitate (Sigma, Shanghai, China). Cells treated with 20 mmol/L mannitol were used as the control group.

Assessment of cell viability
Cell viability was assessed using the Cell Counting Kit-8 (CCK-8, Takara, Dalian, China) according to the manufacturer's instructions. Treated cells (5.5×10 3 cells/well) were cultured with 5% CO 2 at 37°C in a 96-well plate, and each well had 3 replicates. Next, CCK-8 dye was added into each well and incubated for 3 h. Then, the optical density (OD) of each well was measured using a multimode microplate reader (BioTek Instruments, Inc, VT, USA) at 450 nm.

Cell apoptosis assay
Flow cytometry was performed to determine the cell apoptosis rate using an Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) following the manufacturer's instructions. Cultured cells were rinsed 3 times with phosphatebuffered saline (PBS), then the cells were digested with trypsin. The suspended cells (1×10 5 cells) were centrifuged for 5 min at 1000 g and the supernatant was discarded. The cells were gently suspended with 195 μL Annexin V-FITC and 10 μl propidium iodide (PI) and mixed gently. Afterwards, liquids were subjected to flow cytometry (BD, Biosciences, CA, USA). In addition, we used a terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate in situ nick-end labeling (TUNEL) detection kit (Roche, Shanghai, China) to analyze cell apoptosis according to the kit instructions. Treated cells were visualized with a fluorescence microscope after DAPI staining.

Lactate dehydrogenase (LDH) cytotoxicity assay
Cells from different treatment groups incubated in a 96-well plate for 24 h. The plate was removed from the incubator, then LDH release reagent was added into the "maximum enzyme activity control" well. The amount of LDH release reagent was 10% of the original culture volume. It was mixed several times, and then continued to incubate for 12 h after adding LDH release reagent. Next, the plate was centrifuged with a porous plate centrifuge at 400 g for 5 min. There was 120 ml of supernatant in each well, and this was added to each well of a new 96-well plate, then the samples were assessed with a multimode microplate reader (BioTek Instruments, Inc, VT, USA).

Western blot assay
Proteins were extracted from treated NCM460 and DLD-1 cells using RIPA Buffer (TransGen, Beijing, China) after transfection for 36 h with proteinase. The protein samples were separated by SDS-PAGE (polyacrylamide gel electrophoresis), and then transferred to PVDF membranes (Merck Millipore). Next, 5% non-fat milk was used to block the transferred PVDF membrane and incubated with TGR5 (ab72608), Akt (ab8805), p65 (ab16502), and Bcl-2 (ab32124) separately at 4°C overnight. All the transferred membranes were washed extensively and then were incubated with corresponding secondary antibodies (TransGen, Beijing, China) for 1 h at room temperature on the next day. The results are from immunoblotting detection using the ECL immunoblotting kit (Millipore, USA).

Statistical analysis
Each experiment was repeated triplicate, and all the results are shown as mean±standard deviation. Comparisons among multiple groups are shown by one-way analysis of variance followed by Tukey's test. Differences between 2 groups were analyzed by t test. Statistical significance was considered as P£0.05.

Results
Sleeve gastrectomy changed the expression of SNHG5 and TGR5 in blood Blood samples were collected from patients with type II diabetes mellitus before sleeve gastric surgery and at 6 months and 12 months after sleeve gastric surgery. Real-time PCR was performed to detect the expression of SNHG5 and TGR5. The result revealed that the level of SNHG5 before surgery was significantly higher than at 6 months after sleeve gastric surgery, and the level of SNHG5 was lowest at 12 months after surgery ( Figure 1A). However, TGR5 showed the opposite trend, in which the level at 6 months after surgery was significantly e920628-3 higher than before surgery, and reached the highest level among the 3 at 12 months ( Figure 1B).
High-glucose and high-fat culturing caused obvious damage to NCM460 and DLD-1 cells We observed damage characteristics of type II diabetes to NCM460 and DLD-1 cells through high-glucose and high-fat culture. The growth status of NCM460 and DLD-1 cells was measured by CCK-8 assay, flow cytometry, and lactate dehydrogenase cytotoxicity assay. The results showed that culturing in high-glucose and high-fat medium reduced the viability of NCM460 and DLD-1 cells (Figure 2A, 2B), increased the apoptosis rate ( Figure 2C, 2D), and increased the release of LDH ( Figure 2E, 2F).
High-glucose and high-fat culture changed the levels of SNHG5 and TGR5 The growth state of NCM460 and DLD-1 cells was changed by high-glucose and high-fat culture, and we further examined whether SNHG5 and TGR5 also changed. The result of real-time PCR showed that SNHG5 expression was increased ( Figure 3A, 3B) and the expression of TGR5 was decreased ( Figure 3C, 3D) by high-glucose and high-fat culturing, as was the mRNA of Bcl-2 ( Figure 3E, 3F). Western blot assay also confirmed that TGR5, Akt, p65, and Bcl-2 protein levels were decreased by high-glucose and high-fat culturing ( Figure 3G).
Inhibition of SNHG5 reversed the effect of high-glucose and high-fat culture In this study, we inhibited the expression of SNHG5 in NCM460 and DLD-1 cells by using siRNA, and the effect of the newly synthesized siRNA against SNHG5 was verified by real-time PCR ( Figure 4A). There were 3 different processing groups: siRNA control and high-glucose and high-fat, siRNA and mannitol, and siRNA and high-glucose and high-fat. CCK-8 assay showed that siRNA of SNHG5 enhanced the viability of NCM460 and DLD-1 cells, and reversed the decrease in cell viability due to high-glucose and high-fat culture ( Figure 4B, 4C). The results showed that siRNA reduced the apoptosis of NCM460 and DLD-1 cells, and reversed the increase of apoptosis rate caused by high-glucose and high-fat culture ( Figure 4D). LDH secretion resulting from influence of high-glucose and high-fat culture was also reversed by siRNA ( Figure 4E, 4F).

SNHG5 siRNA changed the expression of TGR5 gene in NCM460 and DLD-1
We used real-time PCR and Western blot analysis to detect gene expression in NCM460 and DLD-1 cells cultured in siRNA of SNHG5 or high-glucose and high-fat medium. We detected mRNA levels of TGR5 ( Figure 5A) and Bcl-2 ( Figure 5B) in NCM460 cells and found that the decrease in gene expression due to high glucose and high fat culture was alleviated by siRNA. Western blot analysis showed that the decrease in TGR5, p65, and Bcl-2 protein levels that occurred after damage to NCM460 ( Figure 5C) and DLD-1 ( Figure 5D) cells was reversed by transfection of SNHG5 siRNA.

Discussion
The long-term effects of traditional medical treatment of type II diabetes are not ideal. The results of randomized controlled clinical trials show that metabolic surgery has a significantly better long-term control effect on blood glucose levels in diabetic patients than does medical treatment [8,9], and metabolic surgery has been recommended as a treatment for type II diabetes [10]. Sleeve-type gastrectomy became the most frequently used surgical procedure for diabetes in clinical practice due to its remarkable glucose-control effect, relatively simple operation, and low probability of complications [11,12]. Sleeve gastrectomy directly restructures the gastrointestinal tract and plays a direct role in regulating glucose metabolism. Research has confirmed that obesity and diabetes can be caused by gastrointestinal disorders [13].
As a potential new biological regulator, lncRNA has received extensive attention recently, especially via the emergence of sensitive high-throughput genomic technologies (such as microarray technology and next-generation sequencing technology), bringing unprecedented ability to detect new transcripts. lncRNA SNHG5 is highly expressed in cisplatin-resistant gastric cancer and promotes cisplatin resistance by regulating apoptosisrelated genes and drug resistance-related genes [14]. SNHG5 has also been confirmed to participate in the occurrence and e920628-7 progression of osteosarcoma [15]. The relationship between lncRNA and diabetes and its complications is also constantly being explored, including the role of SNHG5. One study found that in high-glucose cultured mesangial cells (MCs), the expression of PVT1 is increased. After knockdown of PVT1 by siRNA, many important extracellular matrix-related gene expressions, such as FN1, COL4A1, TGFb-1, andPAI-1, were downregulated and slow the progression of diabetes-induced kidney disease [16,17]. Information about new lncRNAs that are upregulated or downregulated in type II diabetes, as well results of as transcriptomics studies, may reveal a new class of non-coding transcripts that further biological understanding.
G-protein-coupled receptor (GPCR) is a superfamily of receptors that play key roles in a variety biological effect pathways. The G protein-coupled bile acid receptor Gpbarl (TGR5) belongs to the GPCR family, and TGR5 is classified as a GPCR and is considered to be a bile acid receptor subclass. In addition to being involved in the digestive process, bile acids also act as regulatory molecules involved in the binding of TGR5 and foresaid X receptor (FXR) in the liver and intestine, activating related signaling pathways, and thereby regulating glucose metabolism, lipid metabolism, and bile gene expression of related enzymes and proteins during acid metabolism [18,19]. In vitro use of free bile acids stimulate the intestinal tract by inhibiting the release of glucagon and stimulating insulin secretion by activating the TGR5 receptor, thereby achieving the goal of lowering blood glucose levels. Activation of the TGR5 pathway by the TGR5 transgenic mouse model significantly changed glucose tolerance in obese mice [20]. In the present study, we assessed whether the regulatory role of lncRNA affects TGR5, then we established a relationship between lncRNA and highglucose-induced cell injury.
In the present study, levels of SNHG5 and TGR5 in peripheral blood showed that SNHG5 decreased after surgery, while TGR5 showed the opposite trend. The level of SHNG5 was elevated while TGR5 was decreased by culturing in high-glucose and high-fat medium. The expressions of TGR5, Akt, p65, and Bcl-2 were also affected by high-glucose and high-fat culturing. Inhibition of the expression of SNHG5 reversed the damage (growth inhibition and increased apoptosis) to cells caused e920628-8 by high-glucose and high-fat culturing. Inhibition of SNHG5 also caused an increase in TGR5 and Bcl-2 expression. When SNHG5 was inhibited, the expression of TGR5 was reversed, which resulted in changes in the expression of downstream genes related to cell growth and apoptosis.

Conclusions
Our study found that SNHG5 is involved in the damage of colorectal cells, mainly induced by high-glucose and high-fat culture conditions, partly through regulation of the TGR5 gene. The main effect on the body's function in type II diabetes mellitus occurs via damage to functional cells. One of the mechanisms underlying the effect of sleeve gastrectomy for type II diabetes mellitus is by regulating the SNHG5/TGR5 pathway and ultimately reducing blood glucose, and thereby reducing the extent of cell injury.

Availability of data and materials
The analyzed datasets generated during the study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The study protocol was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of JiaMusi University.