Yonsei Med J.  2020 Mar;61(3):210-217. 10.3349/ymj.2020.61.3.210.

The MicroRNA hsa-let-7g Promotes Proliferation and Inhibits Apoptosis in Lung Cancer by Targeting HOXB1

• Cui F
• Zhou Q
• Xiao K
• Ma S

• KMID: 2470917
• DOI: http://doi.org/10.3349/ymj.2020.61.3.210

Abstract

PURPOSE
The goal of this study was to explore the effects of hsa-let-7g on cell proliferation and apoptosis, and elucidate its role in lung cancer development.
MATERIALS AND METHODS
The expression levels of has-let-7g and HOXB1 in tissues and cells were measured by qRT-PCR. An inhibitor of hsa-let-7g or one targeting a control messenger RNA were transfected into A549 and H1944 lung cancer cells, and the effects of hsa-let-7g dysregulation on cell viability and apoptosis were analyzed using CCK-8 and apoptosis detection assays. HOXB1 was confirmed as the target gene of hsa-let-7g, based on luciferase reporter assay results. The relationship between hsa-let-7g and HOXB1 was confirmed by co-transfection of inhibitors of hsa-let-7g and HOXB1 followed by Western blot, CCK-8, and apoptosis detection assays.
RESULTS
We observed high expression of hsa-let-7g in lung cancer tissues compared to the corresponding normal tissues, and generally higher expression of hsa-let-7g in patients with advanced tumor classification. The results of CCK-8 and apoptosis detection experiments showed that the inhibition of hsa-let-7g significantly inhibited proliferation of A549 and H1944 cells, but also promoted apoptosis. HOXB1 is a specific target of hsa-let-7g, and downregulation of HOXB1 in lung cancer cells reversed the suppressive effects caused by knocking down hsa-let-7g.
CONCLUSION
These data collectively suggest that the expression of hsa-let-7g inhibits lung cancer cells apoptosis and promotes proliferation by down-regulating HOXB1. The results from this study demonstrate the potential of hsa-let-7g/HOXB1 axis as a therapeutic target for the treatment of lung cancer.

Keyword

Lung cancer; miRNA; hsa-let-7g; HOXB1; proliferation; apoptosis

MeSH Terms

Apoptosis*
Blotting, Western
Cell Proliferation
Cell Survival
Classification
Down-Regulation
Humans
Luciferases
Lung Neoplasms*
Lung*
MicroRNAs*
RNA, Messenger
Sincalide
Luciferases
MicroRNAs
RNA, Messenger
Sincalide

Figure

• 

  Fig. 1 hsa-let-7g levels in lung cancer. (A) The expression of hsa-let-7g in 20 paired normal-lung tumor samples. (B) Correlation of hsa-let-7g expression levels at different tumor stages. (C) HOXB1 expression in 20 paired normal-tumor lung samples. *p<0.05. HOXB1, homeobox B1, mRNA: messenger RNA.

• 

  Fig. 2 Promotion of lung cancer by hsa-let-7g. (A) Expression of hsa-let-7g in A549 cells with a negative control lentivirus (LV-NC) or hsa-let-7g LV-inhibitor. (B) Proliferation in A549 cells after inhibition of has-let-7g expression. (C) Cell apoptosis in A549 cells after inhibition of has-let-7g expression. (D) Expression of hsa-let-7g in A549 cells transfected with LV-NC or hsa-let-7g LV-inhibitor. (E) Expression of hsa-let-7g in H1944 cells with a negative control lentivirus (LV-NC) or hsa-let-7g LV-inhibitor. (F) Proliferation in H1944 cells after inhibition of has-let-7g expression. (G) Cell apoptosis in H1944 cells after inhibition of has-let-7g expression. (H) Expression of hsa-let-7g in H1944 cells transfected with LV-NC or hsa-let-7g LV-inhibitor. *p<0.05.

• 

  Fig. 3 Modulation of HOXB1 by hsa-let-7g. (A, B) Direct targeting of hsa-let-7g on HOXB1 was confirmed by luciferase activity assay. (C, D) Expression level of HOXB1 mRNA in A549 and H1944 cells post-transfection with hsa-let-7g mimics or mimic-NC and LV-inhibitor or negative congtrol lentivirus (LV-NC). (E, F) HOXB1 protein expression level in A549 and H1944 cells transfected with LV-NC or LV-inhibitor. *p<0.05. HOXB1, homeobox B1, mRNA: messenger RNA.

• 

  Fig. 4 Reversion in A549 cells. (A, B) The expression level of HOXB1 was significantly downregulated in A549 cells after co-transfection with LV-sh-HOXB1 and has-let-7g inhibitor, compared to cells transfected with has-let-7g inhibitor or negative congtrol lentivirus (LV-NC). (C) Proliferation increased in A549 cells after co-transfection with LV-sh-HOXB1 and LV-inhibitor, compared to LV-inhibitor. (D) Apoptosis decreased in A549 cells after co-transfection with LV-sh-HOXB1 and LV-inhibitor, compared to LV-inhibitor alone. *p<0.05. HOXB1, homeobox B1.

• 

  Fig. 5 Reversion in H1944 cells. (A, B) The expression level of HOXB1 was significantly downregulated in H1944 cells after co-transfection with LV-sh-HOXB1 and has-let-7g inhibitor, compared to cells transfected with has-let-7g inhibitor or negative congtrol lentivirus (LV-NC). (C) Proliferation increased in H1944 cells after co-transfection with LV-sh-HOXB1 and LV-inhibitor, compared to LV-inhibitor alone. (D) Apoptosis decreased in H1944 cells after co-transfection with LV-sh-HOXB1 and LV-inhibitor, compared to LV-inhibitor alone. *p<0.05. HOXB1, homeobox B1.

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