Yonsei Med J.  2019 May;60(5):414-422. 10.3349/ymj.2019.60.5.414.

MicroRNA-362 Inhibits Cell Proliferation and Invasion by Directly Targeting SIX1 in Colorectal Cancer

• Wan J
• Yang J
• Qiao C
• Sun X
• Di A
• Zhang L
• Wang D
• Zhao G

• KMID: 2443244
• DOI: http://doi.org/10.3349/ymj.2019.60.5.414

Abstract

PURPOSE
Colorectal cancer (CRC) is the third most common cancer in China and poses high morbidity and mortality. In recent years, increasing evidence has indicated that microRNAs played important functions in the occurrence and development of tumors. The purpose of this study was to identify the biological mechanisms of miR-362 in CRC.
MATERIALS AND METHODS
Quantitative real-time PCR was carried out to assess the expression of miR-362 and SIX1. The Kaplan-Meier method was employed to evaluate the 5-year overall survival of CRC patients. The proliferative and invasive abilities of CRC cells were assessed by MTT and transwell assays.
RESULTS
miR-362 was significantly decreased in CRC tissues and cell lines, compared to the normal tissues and normal cells. A significant connection was confirmed between the overall survival of 53 CRC patients and low expression of miR-362. Downregulation of miR-362 inhibited the proliferation and invasion through binding to the 3"²-UTR of SIX1 mRNA in CRC. Additionally, we discovered that SIX1 was a direct target gene of miR-362 and that the expression of miR-362 had a negative connection with SIX1 expression in CRC. SIX1 could reverse partial functions in the proliferation and invasion in CRC cells.
CONCLUSION
miR-362 may be a prognostic marker in CRC and suppress CRC cell proliferation and invasion in part through targeting the 3"²-UTR of SIX1 mRNA. The newly identified miR-362/SIX1 axis provides insight into the progression of CRC.

Keyword

miR-362; SIX; proliferation; invasion; colorectal cancer

MeSH Terms

Cell Line
Cell Proliferation*
China
Colorectal Neoplasms*
Down-Regulation
Humans
Methods
MicroRNAs
Mortality
Real-Time Polymerase Chain Reaction
RNA, Messenger
MicroRNAs
RNA, Messenger

Figure

• 

  Fig. 1 miR-362 expression was low in CRC, and downregulation of miR-362 predicted poor prognosis. (A) The expression of miR-362 was higher in CRC than paracancerous tissues (PT). (B) Kaplan-Meier analysis indicated that miR-362 downregulation predicted poor prognosis in CRC. (C) The expression of miR-362 was lower in LOVO and SW480 CRC cells than in normal CCD-18Co cells. (D) The efficiency of transfected miR-362 mimic or inhibitor was evaluated by qRT-PCR. **p<0.01, ***p<0.001. CRC, colorectal cancer.

• 

  Fig. 2 miR-362 suppressed cell proliferation and invasion. (A) The cell viability was regulated by exogenous changing miR-362 in SW480 cells. (B) Transwell assay revealed miR-362 mimic inhibits cell invasion, while miR-362 inhibitor promotes invasive ability in SW480 cells (0.5% crystal violet, ×200). *p<0.05, **p<0.01, ***p<0.001.

• 

  Fig. 3 SIX1 was upregulated in CRC, and overexpression of SIX1 predicted poor prognosis. (A) The expression of SIX1 reflected significant increases in CRC tissues, compared to paracancerous tissues (PT). (B) miR-362 exhibited negative correlations with the expression of SIX1 in CRC tissues. (C) Upregulation of SIX1 predicted poor 5-year overall survival in CRC patients. (D) The expression of SIX1 was higher in LOVO and SW480 cells, compared with CCD-18Co cells. **p<0.01, ***p<0.001. CRC, colorectal cancer.

• 

  Fig. 4 SIX1 was a target of miR-362, and the expression of SIX1 was mediated by miR-362. (A) TargetScan was employed to predict SIX1 as a potential target gene of miR-362. (B) Luciferase reporter assay was applied to determine whether miR-362 targets SIX1. (C) miR-362 regulated the expression of SIX1. #p>0.05, **p<0.01, ***p<0.001.

• 

  Fig. 5 Knockdown of SIX1 inhibited the proliferation and invasion of SW480 cells. (A) The siRNA-SIX1 was conducted to knockdown SIX1 in SW480 cells. (B) In comparison with siRNA-NC, the cell viability was decreased by transfecting siRNA-SIX1 in SW480 cells. (C) Transwell assay revealed that siRNA-SIX1 reduces the invasive ability in SW480 cells (0.5% crystal violet, ×200). **p<0.01.

• 

  Fig. 6 SIX1 could reverse partial roles of miR-362 on cell proliferation and invasion. (A) RT-qPCR detected the transfection efficiency after re-expressed SIX1 in miR-362 mimic-transfected SW480 cells. Alterations of the proliferative (B) and invasive (C) abilities after re-expressing SIX1 in miR-362 mimic transfected SW480 cells (0.5% crystal violet, ×200). *p<0.05, **p<0.01, ***p<0.001.

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