Spatholobus suberectus extract suppresses proliferation and EMT, and promotes apoptosis in palmitic acid induced vascular endothelial cells by inhibiting LncRNA MALAT1 via VEGF signaling pathway

Purpose: In type 2 diabetes, palmitic acid could damage vessels and induce insulin resistance. This present in vitro study evaluates the possible role of Spatholobus suberectus (FSS) extract in diabetes. Methods: Human HUVECc cells were treated with palmitic acid, palmitic acid and Spatholobus suberectus extract. MALAT1 overexpression plasmid (pcDNA-MALAT1) and blank vector were transfected into the cells using lipofectamine 2000. RT-qPCR assay was used to evaluated the expression changes of lncRNA, VEGFR2 and VEGFA in the cells as well as Epithelial-Mesenchymal Transition (EMT) biomarkers and apoptosis. CCK-8 was used to detect cell viabilities of HUVECs. Expressions of proteins in VEGF signaling pathway were analyzed using Western Blot. Results: LncRNA MALAT1 had high expression in diabetes-like cells and suppressed proliferation and EMT but promoted apoptosis. The SS extract promoted proliferation and EMT and repressed apoptosis in diabetes-like HUVECs cells. The promotion of apoptosis by LncRNA MALAT1, inhibition of apoptosis and regulated functions of diabetes-like HUVECs cells by SS extract occurred via the VEGF signaling pathway Conclusion: SS extract might contribute to survival of cells by inhibiting MALAT1 via VEGF signaling pathway in vitro, suggesting FSF might be a potential therapeutic agent in the treatment of diabetes.


INTRODUCTION
Diabetes is a metabolic disease with damaged functions of pancreatic islet or insulin resistance that may be caused by genetic factors, immune disorders, microbial infection or obesity, etc [1]. According to researches, there are about 0.387 billion people with diabetes all over the world and it is predicted by International Diabetes Federation (IDF) that the numbers will reach 0.592 billion by 2035 [2]. The high morbidity and mortality of cardiovascular diseases caused by diabetes has become a serious problem that threatens human health. Patients with diabetes often have abnormal angiogenesis, which is the main factor of diabetic cardiovascular complications [3]. Vascular endothelial growth factor (VEGF) plays an important role in regulating angiogenesis in physiological and pathological conditions [4]. VEGF and its receptor vascular endothelial factor receptor-2 (VEGFR-2) axis are affected by diabetes to militate growth of cardiovascular diseases [5]. In patients with type 2 diabetes, the level of palmitic acid is significantly increased, which could injure vascular endothelial cells and produce lipotoxicity. Moreover, it can influence to insulin signaling pathway, causing dysfunctions of vascular endothelial cells and leading to insulinresistant state [6].
LncRNA is a kind of RNA which has no proteincoding feature and full-length is longer than 200 nucleotides [11]. It is distributed in cytoplasm, nucleus and cell organelle but mainly in nucleus [12]. It has high tissue specificity, whose expressions are not only different in different tissues, but in same tissues; in different positions or different stages of the same tissue or organ [13,14]. LncRNA-MALAT1 has high expression in many cancer cell lines, which has close connection with cell migration and cancer recurrence [15]. For instance, MALAT1 could be an individual predictor for recurrence of liver cancer, suppression of which could inhibit survival rate and migration of HepG2 cells [16]. However, in diabetes MALAT1 is upregulated and its inhibition can decrease apoptosis of vascular endothelial cells.
Among these compounds, flavones are the main active element as they have broad pharmacological functions including anti-tumor, antibiosis and control diseases of cardiovascular system, blood system, nervous system and atrophic arthritis [9]. S. suberectus has been reported to promote proliferation and differentiation of hemopoietic progenitor in mice to induce hematopoiesis [10]. Thus, there might be a connection between LncRNA MALAT1 and FSS because of the presence of flavones in FSS.
The objective of this study was to evaluate whether Spatholobus suberectus extract suppresses proliferation and EMT, and promotes apoptosis of palmitic acid induced vascular endothelial cells by inhibiting LncRNA MALAT1 via VEGF signaling pathway using human HUVECs. In this study, palmitic acid was applied to induce vascular endothelial cells to build diabetes cellular model. Thereafter, FSS was used for intervention to detect proliferation of vascular endothelial cells and effects on VEGF signaling pathway. Proliferation and apoptosis of HUVECs and proteins in VEGF signaling pathway were analyzed for detecting mechanism of action.

EXPERIMENTAL Preparation of Spatholobus suberectus (SS)
Dried SS was bought from a traditional Chinese drug store and pulverised into powder and a sample of 9.3 g was extracted with 50% ethyl alcohol in water at 50 o C for 120 min twice. Then the extract was evaporated to dryness using a rotary evaporator (RE-52A, Shanghai Yarong Biochemistry, China) and the residue was made up to 100ml with 50% ethyl alcohol for further use.

Cell culture and treatment
Human HUVECs cells (purchased from ATCC, USA) were revived using standard procedure and then cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), 100μg/ml penicillin and streptomycin at 37 o C and 5% CO2. Then, the cells in their log phase were digested with trypsin to make single cell suspension for further experiments which were shared into four parts; Part 1: control (normal) cells (no SS and no palmitic acid treatment) Part 2: diabetes-like cells -600μmol/l palmitic acid was added so as to mimic the diabetes-like cells Part 3: flavone treated diabetes-like cells 1 -600μmol/l palmitic acid and 1.00mg/ml of SS extract were added Part 4: SS treated diabetes-like cells 2 -600μmol/l palmitic acid and 2.00mg/ml of SS extract were added Transfection Adopting the method earlier described [19], each of the cells. types were seeded into 6-well plate and incubated in the DMEM medium until the confluence reached 70%. The sequences of MALAT1 (Thermo Fisher Scientific, China) were amplified using polymerase chain reaction (PCR) and then subcloned into pcDNA3.1 (Thermo Fisher Scientific, China) to produce MALAT1 overexpression plasmid (pcDNA-MALAT1). The resultant pcDNA3.1-MALAT1 and blank vector were transfected into cells using lipofectamine 2000 (Thermo Fisher Scientific, China) according to the manufacturer's instruction. RT-qPCR was done to quantify the efficiency of transfection.

CCK-8 assay
Disease-like cells, extract treated disease-like cells (1mg/ml and 2mg/ml), and oe-MALAT1 extract treated cells (2mg/ml) were selected for cell viability assay. Cells were seeded into 96well plate with 1×10 5 cells per well and then incubated in saturated humidity incubator at 37 C and 5% CO2. CCK-8 (10μl) was added into each well after 24h, 48h and 72h and the optical density (OD) values were determined at 490nm using microplate reader (Thermo Fisher Scientific, China).

Western blot
At 48h after transfection, cells from the above groups were lysed using RIPA buffer (Thermo Fisher, USA) for 15 min on ice and then centrifuged to get supernatant liquid at 12000 rpm, 4 o C for 15 min. Concentrations of proteins was evaluated using BCA protein assay kit (Thermo Fisher, USA) based on manufacturer's instruction to adjust proteins to the same concentrations. Proteins were denatured with Blue Juice™ Gel Loading Buffer (10×) (Invitrogen™, USA) at 95 o C for 8 min and then separated using SDS-PAGE and transferred into PVDF membranes. The membranes were then blocked with 8% skimmed milk powder and primary antibodies added prior to incubation at 4 ℃ overnight. was applied for coloration and gray color of proteins were used to quantify expressions of proteins.

Statistical analysis
Each experiment was done in triplicates, analysed using SPSS 19.0 (IBM, USA) and data were reported as mean ± SD. Comparison of data was achieved using Student's T test as appropriate. P<0.05 was considered significant at 95% confidence interval.

LncRNA MALAT1 had high expression in diabetes-like cells and suppressed proliferation and EMT but promoted apoptosis
When the expressions of lncRNA MALAT1 was evaluated in normal human HUVECs and palmitic acid induced HUVECs, lncRNA MALAT1 expressed higher in diabetes-like HUVECs ( Figure 1A) when compared to normal cells. After MALAT1 was overexpressed, its expression was higher than control group in induced cells ( Figure  1B). The results of CCK-8 showed that overexpressed lncRNA MALAT1 significantly reduced cell viability of induced HUVECs ( Figure  1C). As for EMT, analysis of RNA expression of biomarkers showed that E-cadherin was promoted while N-cadherin and Vimentin were inhibited by up-regulated lncRNA MALAT1 ( Figure 1D-F). Moreover, analysis of the RNA expression of biomarkers related to apoptosis showed that in overexpressed MALAT1 group, expressions of Bcl-2 and Bcl-xL was suppressed while expressions of caspase-3 was promoted ( Figure 1G-I).

Spatholobus suberectus extract promoted proliferation and EMT and repressed apoptosis in diabetes-like HUVECs
With increasing concentrations of S. suberectus extract (0.00mg/ml, 1.00 mg/ml and 2.00mg/ml corresponding to increasing concentration of flavones), viability of HUVECs was increasingly up-regulated (Figure 2A). E-cadherin expression was significantly decreased and expressions of N-cadherin and Vimentin was increased as densities of flavones of S. suberectus upregulated ( Figure 2B-D). RNA expression of apoptosis proteins indicated that expressions of Bcl-2 and Bcl-xL was increased but caspase-3 was repressed with increasing concentrations of flavone ( Figure 2E-G).  In normal human HUVECs and palmitic acid induced human HUVECs, VEGF-A and VEGFR-2 were detected for RNA expression, which showed that VEGF-A and VEGFR-2 were activated in diabetes-like HUVECs compared to normal HUVECs ( Figure 3A-B). RT-qPCR and Western Blot results indicated that up-regulated lncRNA MALAT1 activated VEGF-A and VEGFR-2 in induced cells ( Figure 3C-D). When VEGFR2 suppressor SKLB610 was added, expression of VEGF-A and VEGFR-2 was significantly suppressed (Figure 3E-F). Anti-apoptosis biomarkers Bcl-2 and Bcl-xL were down regulated while caspas-3 was increased by upregulated MALAT1, implying that cell apoptosis was increased by MALAT1 upregulation. However, when SKLB610 was applied together with overexpressed MALAT1, the effects were counteracted in part with increased anti-apoptosis biomarkers and decreased Caspas-3 compared to oe-MALAT1 group, suggesting that MALAT1 might promote cell apoptosis via VEGF signaling pathway ( Figure 3G-H).

Spatholobus suberectus extract inhibits MALAT1 and regulated functions of HUVECs via VEGF signaling pathway
In palmitic acid induced human HUVECs, protein expression of VEGF-A and VEGFR-2were detected by Western Blot ( Figure 4A). With increasing concentrations of S. suberectus extract, VEGF-A and VEGFR-2 expression was both inhibited in a dose-dependent way. Thereafter, lncRNA MALAT1 expression was significantly lower after S. suberectus extract was added into human HUVECs in a dose-dependent manner ( Figure 4B). Moreover, biomarkers related to EMT were detected as well; relative RNA expression of E-cadherin was promoted by overexpression of MALAT1 but the extract inhibited the function of overexpressed MALAT1 ( Figure 4C). Meanwhile, suppressed N-cadherin and Vimentin by up-regulated lncRNA MALAT1 was reversed in the presence of the extract ( Figure 4D-E). Similar results were obtained in apoptosis analysis. Inhibited expression of Bcl-2 and Bcl-xL by overexpressed lncRNA MALAT1 was up-regulated by the extract in a dosagedependent manner. The caspase-3, which was activated in overexpressed lncRNA MALAT1 groups, was inhibited by the extract as well ( Figure 4F).

DISCUSSION
Based on earlier study, catechinic acid of flavone present in S. suberectus may induce activation of GM-CSA, BPA, MK-CSA to increase creation of blood cells and also promote red blood cells and hemoglobin of mice with anemia [20]. LncRNA MALAT1 show higher expression in patients with various complications related to diabetes and it promoted apoptosis in vitro [20,21]. This study has revealed that LncRNA MALAT1 had high expression in diabetes-like cells and suppressed proliferation and EMT but promoted apoptosis. The SS extract promoted proliferation and EMT and suppressed apoptosis but increased cell viability in diabetes-like HUVECs cells. Using RT-qPCR and Western Blot, high expression of VEGFR-2 and VEGF-A in disease-like cells and oe-MALAT1 activated VEGF signaling. With Inactivation of VEGF signaling with the antagonist of VEGF pathway, SKLB610, reduced the cell death indicating that MALAT1 may promote cell death via VEGF signaling pathway.
By connecting SS extract with VEGF and MALAT1 using different dosages of SS extract, it became clear that the proteins in VEGF signaling, VEGFR-2 and VEGF-A, gradually decreased as the dosage of SS extract increased. EMT process and apoptosis were examined and the findings pointed out that overexpressed MALAT1 inhibited the EMT process and promoted apoptosis while SS extract could offset the apoptosis-promoting effect of MALAT1 in vitro, implying that SS extract might protect against cell apoptosis through MALAT1. In conclusion, it was suggested that SS extract might protect the human vascular endothelial cells from injury induced by palmitic acid through inhibiting MALAT1 via VEGF signaling pathway. VEGF was first discovered in ascites and tumor tissues of guinea pig in 1983 [22]. There are five members in VEGF signaling pathway: VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PIGF). VEGFR-2 (KDR/Flk-1) is the primary function receptor which is mainly expressed in vascular endothelial cells [4].It was reported previously that VEGF signaling pathway was activated in glucose-induced HUVECs [23].

CONCLUSION
LncRNA MALAT1 had higher expression in palmitic acid induced HUVECs and regulated cell functions via VEGF signaling. The flavones present in S. suberectus could promote cell viability, EMT and inhibit apoptosis by inhibiting MALAT1 via VEGF signaling pathway suggesting that SS EXTRACT might be a potential remedy for injuries involving blood vessel in diabetes.