Effect of SNHG11/miR-7-5p/PLCB1 Axis on Acute Pancreatitis through Inhibiting p38MAPK Pathway

Figures

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  Figure 1. Successful establishment of a model of AP in Rat and cell lines. (A,B) The establishment of the rat model of acute pancreatitis was confirmed by HE staining, the pathological scoring was performed in two groups, n = 5 in each group, and representative images have been shown. (C,D) Apoptosis of pancreatic tissues in rats evaluated by TUNEL staining (n = 5). (E,F) Serum levels of AMY and LIPA of rats examined by ELISA (n = 5). (G–I) Expression of IL-6, TNF-α, and IL-1β in the rats, AR42J, and HPDE6-C7 assessed by ELISA (n = 3). The data are presented as means ± SDs, * p < 0.05; ** p < 0.01; *** p < 0.001, between the indicated groups.

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  Figure 2. Pancreatitis is accompanied by a decrease in lncRNA-SNHG11. (A,B) The matrix (A) and volcano (B) maps of differential lncRNA obtained by RNA-seq in AP vs. sham. (C) The CTD database was used to screen pancreatitis-related genes and intersect with different lncRNAs to find the common gene SNHG11. (D–F) Expression of SNHG11 in the rats, AR42J and HPDE6-C7 assessed by qPCR (n = 3). (G,H) The stable overexpression strain of SNHG11 was constructed in AR42J and HPDE6-C7 cells, and the transfection effect was verified by qPCR experiment (n = 3). (I,J) The effect of SNHG11 on cell death induced by caerulein was observed by Trypan blue test in AR42J and HPDE6-C7(n = 3). (K,L) Cell proliferation was assessed by CCK8 assay in AR42J and HPDE6-C7 cell lines (n = 3). (M–R). Expression of IL-6, TNF-α, and IL-1β in the AR42J and HPDE6-C7 cell lines (empty vs. SNHG11) assessed by ELISA (n = 3). The data are presented as means ± SDs, * p < 0.05; ** p < 0.01; *** p < 0.001, between the indicated groups. ns = no significance.

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  Figure 3. MiR-7-5p binds to lncRNA-SNHG11 in pancreatic cells. (A) Prediction of potentially downstream miRNAs bound to SNHG11 by Annolnc, Encori, and Diana databases. (B) Prediction of the binding position of SNHG11 and miR-7-5p through the secondary structure of SNHG11. (C) Schematic diagram of the MUT-SNHG11 plasmid. (D–G) The relationship between SNHG11 and miR-7-5p was verified by dual-luciferase reporter assay and RIP assay in AR42J and HPDE6-c7 cells. (H–J) The expression of miR-7-5p was assessed in rat tissues, AR42J, and HPDE6-C7 cells. The data are presented as means ± SDs, n = 3 for each group, * p < 0.05; ** p < 0.01; *** p < 0.001, ns = no significance between the indicated groups.

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  Figure 4. LncRNA-SNHG11 plays a role in pancreatitis through MiR-7-5p. (A,B) The regulation effect of SNHG11 expression on MiR-7-5p was observed in AR42J and HPDE6-C7 cells. (C,D) The expression of miR-7-5p in different pancreatitis cell models was verified. (E,F) Cell proliferation was assessed by CCK8 assay in AR42J and Hpde6-C7 Cell models. (G–J) The effects of SNHG11 on cell death and apoptosis in AR42J and HPDE6-C7 cell models were observed by Trypan blue assay and Flow cytometry assay. (K,L) Expression of IL-6, TNF-α, and IL-1β in the AR42J and HPDE6-C7 cell lines assessed by ELISA. The data are presented as means ± SDs, n = 3 for each group, * p < 0.05; ** p < 0.01; *** p < 0.001, NS = no significance, between the indicated groups.

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  Figure 5. MiR-7-5p binds to PLCB-1 and regulates its expression in pancreatitis. (A) The potential target mRNAs of miR-7-5p were predicted in a public database and intersected with RNA-seq data to find AP-related genes. (B–G) The expression of PLCB1 was explored in rat tissues, AR42J, and HPDE6-C7 cells by Q-PCR assay and WB assay. (H) Schematic diagram of the PLCB1-WT and PLCB1-Mut luciferase vectors. (I,J) Relative luciferase activities in AR42J and HPDE6-C7 cells co-transfected with PLCB1-WT or PLCB1-Mut and the miR-7-5p mimic, inhibitor, or corresponding negative control. (K,L) The interaction between miR-7-5p and PLCB1 was confirmed by RIP assay in AR42J and HPDE6-C7 cells. (M–R) The regulation effect of MiR-7-5p expression on PLCB1 was observed by qPCR assay and WB assay in AR42J and HPDE6-C7 cells. The data are presented as means ± SDs, n = 3 for each group, * p < 0.05; ** p < 0.01; *** p < 0.001, ns = no significance between the indicated groups.

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  Figure 6. MiR-7-5p plays a promoting role through PLCB-1 in pancreatitis. (A–F) The expression of PLCB1 was verified in different cell models. (G,H) Cell proliferation was assessed by CCK8 assay in Figure 6A–F cell models. (I,J) Cell apoptosis was monitored by flow cytometry assay in Figure 6A–F cell models. (K,L) The effects of SNHG11 on cell death caused in Figure 6A–F cell models were observed by Trypan blue assay. (M,N) Expression of IL-6, TNF-α, and IL-1β in Figure 6A–F cell models were assessed by ELISA. The data are presented as means ± SDs, n = 3 for each group, * p < 0.05; ** p < 0.01; *** p < 0.001, NS = no significance between the indicated groups.

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  Figure 7. SNHG11/miR-7-5p/PLCB1 plays an inhibitory role in pancreatitis by inhibiting the p38MAPK signaling pathway. (A) The bubble chart shows a significant enrichment of the KEGG pathway by PLCB1-related ceRNA genes. (B–E) In AR42J and HPDE6-C7 cells, the high expression of SNHG11 and PLCB1 could inhibit the expression of p-P38MAPK and the activation of the MAPK signaling pathway, while the low expression of MiR-7-5p showed the same effect. (F,G) The effect of SNHG11 on rat models of AP was confirmed by HE staining, and representative images have been shown. (H,I) The expression of SNHG11 and miR-7-5p was assessed in different rat tissues(AP+empty vs. AP+SNHG11). (J–L) The expression of PLCB1 and p-P38MAPK was verified in different rat tissues (AP+empty vs. AP+SNHG11). The data are presented as means ± SDs, n = 3 each group, * p < 0.05; ** p< 0.01; between the indicated groups. (M) Schematic diagram of SNHG11/MiR-7-5p/PLCB1 affecting pancreatitis through activation of p38MAPK pathway.