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. 2021 Jan;20(2):236-246.
doi: 10.1080/15384101.2020.1868161. Epub 2021 Jan 11.

HOXC-AS2 mediates the proliferation, apoptosis, and migration of non-small cell lung cancer by combining with HOXC13 gene

Bin Liu  1   2 , Jing Li  3 , Ji-Man Li  4 , Guang-Yuan Liu  5 , Yong-Sheng Wang  1
Affiliations

HOXC-AS2 mediates the proliferation, apoptosis, and migration of non-small cell lung cancer by combining with HOXC13 gene

Bin Liu et al. Cell Cycle. 2021 Jan.

Abstract

Non-small cell lung cancer (NSCLC) is the highest incidence and mortality of malignant tumors worldwide and has become a global public health problem. Long non-coding RNAs (LncRNAs) are expected to participate in the progression of NSCLC. This study aims to explore the effects and underlying mechanisms of LncRNA HOXC-AS2 on NSCLC cell proliferation, apoptosis, and migration. The Cell Counting Kit-8 (CCK-8) and clone formation assay were used to measure the A549 and HCC827 cell proliferation. The cell apoptosis and migration was respectively analyzed by flow cytometry and transwell assay. RNA immunoprecipitation (RIP) was used to detect the interaction between HOXC-AS2 and HOXC13. The expression of β-catenin, α-SMA, MMP-1, MMP-2 expression, E-cadherin, and Ki-67 expression were determined by Western blot or immunohistochemistry (IHC) assay. We found that HOXC-AS2 was significantly up-regulated in NSCLC tissues. Knockdown of HOXC-AS2 expression resulted in significant decreases in NSCLC cell proliferation, migration, and epithelial-mesenchymal transition (EMT) process marker proteins, simultaneously activated A549 and HCC827 cell apoptosis. RIP assay suggested that HOXC13 was a functional target for HOXC-AS2. And HOXC-AS2 and HOXC13 could positively regulate each other. Compared with the normal tissues, the mRNA level of HOXC13 was increased in NSCLC tissues. HOXC13 silencing counteracted increases of A549 and HCC827 cell proliferation and migration, as well as a decrease of cell apoptosis induced by HOXC-AS2 overexpression. Moreover, HOXC-AS2 silencing reduced tumor growth rate and Ki-67 expression in vivo. Taken together, HOXC-AS2 knockdown inhibited NSCLC cell proliferation and migration, as well as stimulated NSCLC cell apoptosis through regulation of HOXC13 expression.

Keywords: HOXC-AS2; HOXC13; Non-small cell lung cancer.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1.
Figure 1.
HOXC-AS2 is upregulated in NSCLC tissues. (a) Heat map of the dysregulated expression of the top 10 lncRNAs. (AB) The expression of HOXC-AS2 in human NSCLC tissues (n = 20) and normal tissues (n = 10) was compared by RT-qPCR analysis. (B, C, D, and EC, D, E, and F) HOXC-AS2 mRNA expression in A549 and HCC827 cell lines were detected by RT-qPCR analysis. *P < 0.05 (vs normal tissues/si-NC/NC-Vector), **P < 0.01 (vs NC-Vector). Data were represented as means ± standard deviation (SD). Each experiment was performed in triplicate
Figure 2.
Figure 2.
HOXC-AS2 regulates NSCLC cell proliferation, apoptosis, and migration. (a and b) The proliferation of A549 and HCC827 cells were evaluated by CCK-8 and clone formation assay. (c) The apoptosis of A549 and HCC827 cells was assessed by Annexin V FITC/PI staining flow cytometry. (d) The migration ability of SW480 and HT29 cells were evaluated by transwell assay. (e) The β-catenin, α-SMA, MMP-1, MMP-2, and E-cadherin expression in A549 and HCC827 cells was determined by Western blot analysis. β-actin is a loading control. *P < 0.05 (vs si-NC/NC-Vector), **P < 0.01 (vs si-NC/NC-Vector), ***P < 0.001 (vs si-NC/NC-Vector). Data were represented as means ± standard deviation (SD). Each experiment was performed in triplicate
Figure 3.
Figure 3.
HOXC13 is a target gene of HOXC-AS2. (a) The predicted target genes of the dysregulated 10 lncRNAs. The dysregulated lncRNAs were as the center marked with red and the predicted target genes were marked with yellow. (AB) The expression of HOXC13 in human NSCLC tissues (n = 20) and normal tissues (n = 10) was compared by RT-qPCR analysis. (BC) RNA immunoprecipitation (RIP) assay was performed to recover the interaction of HOXC13 and HOXC-AS2. ***P < 0.001 (vs IgG). (C, D, and ED-G) The expression of HOXC13 in A549 and HCC827 cells was tested by Western blot and RT-qPCR analysis. β-actin is a loading control. (FH) The expression of HOXC-AS2 in A549 and HCC827 cells were tested via RT-qPCR analysis. *P < 0.05 (vs normal tissues/si-NC/NC-Vector), **P < 0.01 (vs si-NC/NC-Vector), ##P < 0.01(vs pcDNA-HOXC-AS2+ si-NC). Data were represented as means ± standard deviation (SD). Each experiment was performed in triplicate
Figure 4.
Figure 4.
HOXC-AS2 regulates NSCLC cell proliferation, apoptosis, and migration via activation of HOXC13. (a and b) The proliferation of A549 and HCC827 cells were evaluated by CCK-8 and clone formation assay. (c) The apoptosis of A549 and HCC827 cells was assessed by Annexin V FITC/PI staining flow cytometry. (d) The migration ability of SW480 and HT29 cells were evaluated by transwell assay. (e) The β-catenin, α-SMA, MMP-1, MMP-2, and E-cadherin expression in A549 and HCC827 cells was determined by Western blot analysis. β-actin is a loading control. *P < 0.05 (vs si-NC), **P < 0.01 (vs si-NC), #P < 0.05(vs pcDNA-HOXC-AS2+ si-NC), ##P < 0.01(vs pcDNA-HOXC-AS2+ si-NC). Data were represented as means ± standard deviation (SD). Each experiment was performed in triplicate
Figure 5.
Figure 5.
HOXC-AS2 silencing suppresses NSCLC development in vivo. (a) Compared with the shRNA-NC group, shRNA-HOXC-AS2 inhibited the tumor volume (bar = 10 mm). (b) The expression of Ki-67 was measured using immunohistochemistry (IHC) assay. *P < 0.05 (vs shRNA-NC), **P < 0.01 (vs shRNA-NC). Data were represented as means ± standard deviation (SD). Each experiment was performed in triplicate
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This work was supported by the National Major Science and Technology Projects of China [2017ZX09304023].

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