MiR-129-5p inhibits proliferation of gastric cancer cells through targeted inhibition on HMGB1 expression
Authors: J Guo
B-F Chai
J-P Wang
J Feng
Journal: European review for medical and pharmacological sciences (2020)
Discipline: PHARMACOLOGY & TOXICOLOGY
Keywords: miR-129-5p; gastric cancer cells; proliferation; apoptosis; HMGB1
Affiliations: Institute of Loess Plateau, Shanxi University, Taiyuan, Shanxi, China. .

Abstract:  Objective: The aim of this study was to investigate the effects of micro ribonucleic acid (miR)-129-5p on the proliferation and apoptosis of gastric cancer cells via targeted repression on the expression of high mobility group protein B1 (HMGB1). Patients and methods: Expression levels of miR-129-5p and HMGB1 in gastric cancer tissues (n=25) and adjacent normal tissues were measured via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The regulatory effect of miR-129-5p on the proliferation of gastric cancer MGC-803 and SGC7901 cells was determined through Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed to analyze the apoptosis rate of gastric cancer cells. To further discover the mechanism of miR-129-5p in regulating malignant behaviors of gastric cancer cells, the miRDB database was employed to predict the binding targets of miR-129-5p. Finally, binding sites of HMGB1 3'-untranslated region (3'-UTR) to miR-129-5p were discovered. Subsequently, HMGB1 wild-type or mutant 3'-UTR Luciferase reporter vectors were constructed, and transfected to MGC-803 and SGC7901 cells together with miR-129-5p or negative control miRNA. Next, Western blotting was adopted to measure the protein expression level of HMGB1 in MGC-803 and SGC7901 cells transfected with miR-129-5p or negative control miRNA, so as to investigate whether miR-129-5p affected HMGB1 protein expression. Additionally, to determine whether HMGB1 mediated the regulatory effect of miR-129-5p on the proliferation of gastric cancer cells, MGC-803 and SGC7901 cells were transfected with pcDNA-HMGB1 or pcDNA-vector, respectively. The expression level of HMGB1 was measured via RT-qPCR, and cell proliferation was determined by CCK-8 assay. Results: The expression level of miR-129-5p in gastric cancer tissues was significantly lower than that in adjacent normal tissues (p&lt;0.001). Meanwhile, the level of miR-129-5p was overtly lower in gastric cancer MGC-803 and SGC7901 cell lines than that in normal gastric mucosal epithelial GES-1 cells (p&lt;0.001). These results indicated that miR-129-5p was lowly expressed in gastric cancer tissues and cell lines. Subsequent results demonstrated that the expression of HMGB1 increased remarkably in gastric cancer tissues compared with normal adjacent tissues (p&lt;0.05). The proliferation ability of MGC-803 (p&lt;0.001) and SGC7901 (p&lt;0.01) cells with over-expressed miR-129-5p was remarkably weakened. Overexpression of miR-129-5p distinctly promoted the apoptosis rate of gastric cancer MGC-803 (p&lt;0.01) and SGC7901 (p&lt;0.001) cells. Moreover, miR-129-5p up-regulation significantly reduced the Luciferase activity of wild-type HMGB1 (p&lt;0.001). However, no significant effect was observed on that of mutant HMGB1. The results suggested that overexpression of miR-129-5p significantly down-regulated the level of HMGB1 in gastric cancer cells. In addition, the messenger RNA (mRNA) level of HMGB1 in cells transfected with miR-129-5p also decreased significantly (p&lt;0.001). HMGB1 overexpression overtly reversed the inhibitory effect of miR-129-5p on the proliferation of gastric cancer cells (p&lt;0.05). All these results demonstrated that the miR-129-5p/HMGB1 axis played a key role in regulating the growth of gastric cancer cells. Conclusions: MiR-129-5p suppresses the progression of gastric cancer through targeted inhibition on the expression of HMGB1. 

Retracted: No

PubPeer comments:
 Fig 1A:  A, Expression level of miR-129-5 in gastric cancer tissues and adjacent normal tissues . Fig 2:  Expression level of HMGB1 in gastric cancer tissues and adjacent normal tissues.  Fig 3:  MiR-129-5p expression is negatively correlated with HMGB1 expression in gastric cancer tissues. I am unable to reconcile the range of miR-129-5p and HMGB1 values plotted as swarm plots in Figs 1A and 2, with the scatterplot of Fig 3. Below, the swarm plots lined up with the horizontal and vertical axes of Fig 3. ![file] The text describes collecting  A total of 50 pairs of gastric cancer tissues and adjacent normal tissues . Presumably this is a misprint for  25 pairs . ,