Down-regulation of miRNA-196b expression inhibits the proliferation, migration and invasiveness of HepG2 cells while promoting their apoptosis via the PI3K/Akt signaling pathway

Fanggui Xu; Feng Zhu; Wulin Wang; Wenjie Gao; Xin Chen; Chunzhao Yu

doi:10.14715/cmb/2020.66.3.25

Issue

Vol. 66 No. 3: Issue 3

Issue Published : June 8, 2020

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Down-regulation of miRNA-196b expression inhibits the proliferation, migration and invasiveness of HepG2 cells while promoting their apoptosis via the PI3K/Akt signaling pathway

https://doi.org/10.14715/cmb/2020.66.3.25

Fanggui Xu

Department of General Surgery, Sir Run Run Hospital, Nanjing Medical University, Nanjing, Jiangsu 211166, P.R. China

Feng Zhu

Department of General Surgery, Sir Run Run Hospital, Nanjing Medical University, Nanjing, Jiangsu 211166, P.R. China

Wulin Wang

Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China

Wenjie Gao

Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China

Xin Chen

Department of General Surgery, Sir Run Run Hospital, Nanjing Medical University, Nanjing, Jiangsu 211166, P.R. China

Chunzhao Yu

Department of General Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China

Corresponding Author(s) : Chunzhao Yu

gxse92@163.com

Cellular and Molecular Biology, Vol. 66 No. 3: Issue 3
Article Published : June 5, 2020

Abstract

The purpose of this study was to investigate the effects of microRNA-196b (miRNA-196b) on proliferation, migration, invasiveness and apoptosis of hepatocellular carcinoma cell line (HepG2), and the mechanism involved. MiRNA-196b inhibitor or negative control were transfected into HepG2 cells, while empty liposome vector was used as normal control. The results of transfection were assessed using real-time quantitative polymerase chain reaction (qRT-PCR). Cell proliferation, migration, invasiveness and apoptosis were determined using cell counting kit 8 (CCK-8), scratch test, Transwell invasion assay, and flow cytometric analysis, respectively. The expressions of PIK3, Akt and p-Akt proteins were determined using Western blotting. The HepG2 cells were also treated with PI3K/Akt signaling pathway inhibitor LY294002, and its effect on cell proliferation, migration, invasion, and apoptosis, and expressions of PIK3, Akt, and p-Akt proteins were determined. The results of RT-PCR showed that the relative expression of miRNA-196b in the inhibitor group (0.42 ± 0.13) was significantly lower than that in the blank control group (0.96 ± 0.10) and the negative control group (1.01 ± 0.32) (p < 0.05). The miRNA-196b inhibitor significantly and time-dependently reduced the invasiveness, proliferation migration abilities of HepG2 cells, while promoting their apoptosis (p < 0.05). The expressions of PIK3 and p-Akt proteins were significantly down-regulated in the inhibitor group, when compared with normal and negative control groups (p < 0.05). However, there were no significant differences in the expression of Akt protein among the groups (p > 0.05). After treatment of HepG2 cells with PI3K/Akt signaling pathway inhibitor LY294002, the proliferative, migratory and invasive abilities of cells in the treatment group were significantly enhanced, while cell apoptosis was significantly reduced (p < 0.05). Similarly, the protein expressions of PIK3 and p-Akt in the non-treatment group was significantly upregulated, relative to the treatment group (p < 0.05), but there was no significant difference in the expression of Akt protein between the two groups (p > 0.05). Downregulation of miRNA-196b expression inhibits the proliferation, migration and invasiveness of HepG2 cells, while promoting their apoptosis via a mechanism involving the PI3K/Akt signaling pathway.

Keywords

Liver cancer MiRNA-196b PI3K/Akt signaling pathway Proliferation Apoptosis.

Xu, F., Zhu, F., Wang, W., Gao, W., Chen, X., & Yu, C. (2020). Down-regulation of miRNA-196b expression inhibits the proliferation, migration and invasiveness of HepG2 cells while promoting their apoptosis via the PI3K/Akt signaling pathway. Cellular and Molecular Biology, 66(3), 159–164. https://doi.org/10.14715/cmb/2020.66.3.25

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References

da Motta-Girardi D, Correa TS, Crosara-Teixeira M, Dos Santos Fernandes G. Hepatocellular carcinoma: review of targeted and immune therapies. J Gastrointest Cancer 2018; 49: 227-236.
Masood N, Basharat Z, Khan T, Yasmin A. Entangling relation of micro RNA-let7, miRNA-200 and miRNA-125 with various cancers. Pathol Oncol Res 2017; 23: 707-715.
Shen YN, Bae IS, Park GH, Choi HS, Lee KH, Kim SH. MicroRNA-196b enhances the radio-sensitivity of SNU-638 gastric cancer cells by targeting RAD23B. Biomed Pharmacother 2018; 105: 362-369.
Mo JS, Park YR, Chae SC. MicroRNA 196B Regulates HOXA5, HOXB6 and GLTP expression levels in colorectal cancer cells. Pathol Oncol Res 2018; 12: 1-8.
Nejati-Azar A, Alivand MR. miRNA 196a2 (rs11614913) & 146a (rs2910164) polymorphisms & breast cancer risk for women in an Iranian population. Per Med 2018; 15: 279-289.
Kanno S, Nosho K, Ishigami K, Yamamoto I, Koide H, Kurihara H, et al. MicroRNA-196b is an independent prognostic biomarker in patients with pancreatic cancer. Carcinogenesis 2017; 38: 425-431.
Yu Z, Lin X, Tian M, Chang W. MicroRNA-196b promotes cell migration and invasion by targeting FOXP2 in hepatocellular carcinoma. Oncol Rep 2018; 39: 731-738.
Díaz-Serrano A, Angulo B, Dominguez C, Pazo-Cid R, Salud A, Jiménez-Fonseca P, et al. Genomic profiling of HER2-positive gastric cancer: PI3K/Akt/mTOR pathway as predictor of outcomes in HER2-positive advanced gastric cancer treated with trastuzumab. Oncologist 2018; 23: 1092-1102.
Zhou R, Chen H, Chen J, Chen X, Wen Y, Xu L. Extract from Astragalus membranaceus inhibit breast cancer cells proliferation via PI3K/AKT/mTOR signaling pathway. BMC Complement Altern Med 2018; 18: 83-89.
Fu X, Liu M, Qu S, Ma J, Zhang Y, Shi T. Exosomal microRNA-32-5p induces multidrug resistance in hepatocellular carcinoma via the PI3K/Akt pathway. J Exp Clin Cancer Res 2018; 31: 52-59.
Guerriero I, D'Angelo D, Pallante P, Santos M, Scrima M, Malanga D, et al. Analysis of miRNA profiles identified miR-196a as a crucial mediator of aberrant PI3K/AKT signaling in lung cancer cells. Oncotarget 2017; 8: 19172-19191.
Hartke J, Johnson M, Ghabril M, Manns MP, Greten TF. The diagnosis and treatment of hepatocellular carcinoma. Semin Diagn Pathol 2017; 34: 153-159.
Sartorius K, Sartorius B, Winkler C, Chuturgoon A, Makarova J. The biological and diagnostic role of miRNA's in hepatocellular carcinoma. Front Biosci 2018; 23: 1701-1720.
Qadir MI, Rizvi SZ. MiRNA in Hepatocellular Carcinoma: Pathogenesis and Therapeutic Approaches. Crit Rev Eukaryot Gene Expr 2017; 27: 355-361.
Xu X, Ling Q, Wang J, Xie H, Wei X, Lu D, et al. Donor miR-196a-2 polymorphism is associated with hepatocellular carcinoma recurrence after liver transplantation in a Han Chinese population. Int J Cancer 2016; 138: 620-629.
Li XD, Li ZG, Song XX, Liu CF. A variant in microRNA-196a2 is associated with susceptibility to hepatocellular carcinoma in Chinese patients with cirrhosis. Pathology 2010; 42: 669-673.
Shen J, Wang S, Zhang YJ, Kappil MA, Chen WH, Kibriya MG, et al. Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma. Epigenetics 2012; 7: 1230-1237.
Rebucci M, Sermeus A, Leonard E, Delaive E, Dieu M, Fransolet M, et al. MiRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1. Mol Cancer 2015; 14: 79-83.
Zhang C, An R, Bao YW, Meng XM, Wang TQ, Sun HN, et al. Inhibitory effects of octreotide on the progression of hepatic fibrosis via the regulation of Bcl-2/Bax and PI3K/AKT signaling pathways. Int Immunopharmacol 2019; 73: 515-526.
Fresno-Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, González-Barón M. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 2004; 30: 193-204.
Li NA, Wang W, Xu B, Gong H. MiRNA-196b regulates gastric cancer cell proliferation and invasion via PI3K/AKT/mTOR signaling pathway. Oncol Lett 2016; 11: 1745-1749.

References

da Motta-Girardi D, Correa TS, Crosara-Teixeira M, Dos Santos Fernandes G. Hepatocellular carcinoma: review of targeted and immune therapies. J Gastrointest Cancer 2018; 49: 227-236.

Masood N, Basharat Z, Khan T, Yasmin A. Entangling relation of micro RNA-let7, miRNA-200 and miRNA-125 with various cancers. Pathol Oncol Res 2017; 23: 707-715.

Shen YN, Bae IS, Park GH, Choi HS, Lee KH, Kim SH. MicroRNA-196b enhances the radio-sensitivity of SNU-638 gastric cancer cells by targeting RAD23B. Biomed Pharmacother 2018; 105: 362-369.

Mo JS, Park YR, Chae SC. MicroRNA 196B Regulates HOXA5, HOXB6 and GLTP expression levels in colorectal cancer cells. Pathol Oncol Res 2018; 12: 1-8.

Nejati-Azar A, Alivand MR. miRNA 196a2 (rs11614913) & 146a (rs2910164) polymorphisms & breast cancer risk for women in an Iranian population. Per Med 2018; 15: 279-289.

Kanno S, Nosho K, Ishigami K, Yamamoto I, Koide H, Kurihara H, et al. MicroRNA-196b is an independent prognostic biomarker in patients with pancreatic cancer. Carcinogenesis 2017; 38: 425-431.

Yu Z, Lin X, Tian M, Chang W. MicroRNA-196b promotes cell migration and invasion by targeting FOXP2 in hepatocellular carcinoma. Oncol Rep 2018; 39: 731-738.

Díaz-Serrano A, Angulo B, Dominguez C, Pazo-Cid R, Salud A, Jiménez-Fonseca P, et al. Genomic profiling of HER2-positive gastric cancer: PI3K/Akt/mTOR pathway as predictor of outcomes in HER2-positive advanced gastric cancer treated with trastuzumab. Oncologist 2018; 23: 1092-1102.

Zhou R, Chen H, Chen J, Chen X, Wen Y, Xu L. Extract from Astragalus membranaceus inhibit breast cancer cells proliferation via PI3K/AKT/mTOR signaling pathway. BMC Complement Altern Med 2018; 18: 83-89.

Fu X, Liu M, Qu S, Ma J, Zhang Y, Shi T. Exosomal microRNA-32-5p induces multidrug resistance in hepatocellular carcinoma via the PI3K/Akt pathway. J Exp Clin Cancer Res 2018; 31: 52-59.

Guerriero I, D'Angelo D, Pallante P, Santos M, Scrima M, Malanga D, et al. Analysis of miRNA profiles identified miR-196a as a crucial mediator of aberrant PI3K/AKT signaling in lung cancer cells. Oncotarget 2017; 8: 19172-19191.

Hartke J, Johnson M, Ghabril M, Manns MP, Greten TF. The diagnosis and treatment of hepatocellular carcinoma. Semin Diagn Pathol 2017; 34: 153-159.

Sartorius K, Sartorius B, Winkler C, Chuturgoon A, Makarova J. The biological and diagnostic role of miRNA's in hepatocellular carcinoma. Front Biosci 2018; 23: 1701-1720.

Qadir MI, Rizvi SZ. MiRNA in Hepatocellular Carcinoma: Pathogenesis and Therapeutic Approaches. Crit Rev Eukaryot Gene Expr 2017; 27: 355-361.

Xu X, Ling Q, Wang J, Xie H, Wei X, Lu D, et al. Donor miR-196a-2 polymorphism is associated with hepatocellular carcinoma recurrence after liver transplantation in a Han Chinese population. Int J Cancer 2016; 138: 620-629.

Li XD, Li ZG, Song XX, Liu CF. A variant in microRNA-196a2 is associated with susceptibility to hepatocellular carcinoma in Chinese patients with cirrhosis. Pathology 2010; 42: 669-673.

Shen J, Wang S, Zhang YJ, Kappil MA, Chen WH, Kibriya MG, et al. Genome-wide aberrant DNA methylation of microRNA host genes in hepatocellular carcinoma. Epigenetics 2012; 7: 1230-1237.

Rebucci M, Sermeus A, Leonard E, Delaive E, Dieu M, Fransolet M, et al. MiRNA-196b inhibits cell proliferation and induces apoptosis in HepG2 cells by targeting IGF2BP1. Mol Cancer 2015; 14: 79-83.

Zhang C, An R, Bao YW, Meng XM, Wang TQ, Sun HN, et al. Inhibitory effects of octreotide on the progression of hepatic fibrosis via the regulation of Bcl-2/Bax and PI3K/AKT signaling pathways. Int Immunopharmacol 2019; 73: 515-526.

Fresno-Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, González-Barón M. PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 2004; 30: 193-204.

Li NA, Wang W, Xu B, Gong H. MiRNA-196b regulates gastric cancer cell proliferation and invasion via PI3K/AKT/mTOR signaling pathway. Oncol Lett 2016; 11: 1745-1749.

Author biographies is not available.