Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan 23;11(3):378.
doi: 10.3390/cells11030378.

LncCDH5-3:3 Regulates Apoptosis, Proliferation, and Aggressiveness in Human Lung Cancer Cells

Konrad Kwaśniak  1 , Justyna Czarnik-Kwaśniak  1 , Khrystyna Malysheva  1   2   3 , Katarzyna Pogoda  1 , Olexandr Korchynskyi  1   2   3   4 , Paweł Rybojad  5 , Bożenna Karczmarek-Borowska  6 , Jacek Tabarkiewicz  1   2
Affiliations

LncCDH5-3:3 Regulates Apoptosis, Proliferation, and Aggressiveness in Human Lung Cancer Cells

Konrad Kwaśniak et al. Cells. .

Abstract

(1) Lung cancer (both small cell and non-small cell) is the leading cause of new deaths associated with cancers globally in men and women. Long noncoding RNAs (lncRNAs) are associated with tumorigenesis in different types of tumors, including lung cancer. Herein, we discuss: (1) An examination of the expression profile of lncCDH5-3:3 in non-small cell lung cancer (NSCLC), and an evaluation of its functional role in lung cancer development and progression using in vitro models; (2) A quantitative real-time polymerase chain reaction assay that confirms lncCDH5-3:3 expression in tumor samples resected from 20 NSCLC patients, and that shows its statistically higher expression levels at stage III NSCLC, compared to stages I and II. Moreover, knockout (KO) and overexpression, as well as molecular and biochemical techniques, were used to investigate the biological functions of lncCDH5-3:3 in NSCLC cells, with a focus on the cells' proliferation and migration; (3) The finding that lncCDH5-3:3 silencing promotes apoptosis and probably regulates the cell cycle and E-cadherin expression in adenocarcinoma cell lines. In comparison, lncCDH5-3:3 overexpression increases the expression levels of proliferation and epithelial-to-mesenchymal transition markers, such as EpCAM, Akt, and ERK1/2; however, at the same time, it also stimulates the expression of E-cadherin, which conversely inhibits the mobility capabilities of lung cancer cells; (4) The results of this study, which provide important insights into the role of lncRNAs in lung cancer. Our study shows that lncCDH5-3:3 affects important features of lung cancer cells, such as their viability and motility. The results support the idea that lncCDH5-3:3 is probably involved in the oncogenesis of NSCLC through the regulation of apoptosis and tumor cell metastasis formation.

Keywords: apoptosis; lncCDH5-3:3; migration; non-small cell lung cancer; proliferation; tumor cell aggressiveness.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Relative expressions (2−ΔCt) of lncCDH5-3:3 in tumor samples from patients with NSCLC in stages I, II, and III of the disease. The diagnosis of NSCLC was histologically confirmed, and the disease stages were categorized from I–III (adenocarcinoma: I-n = 3, II-n = 4, III-n = 3; squamous cell carcinoma: I-n = 3, II-n = 3, III-n = 4) The data are presented as medians and minimum and maximum values. The p-values were calculated on the basis of the Friedman ANOVA and post hoc Dunn’s comparison tests. All statistically significant differences are marked: * p < 0.05. In this figure, the minimum values are marked as formula image; the maximum values as formula image; the median as —; and formula image represents the p-value from the post hoc test. The circles, squares, and triangles filled with the red color represent squamous cell carcinoma samples, while the black circles, squares, and triangles mark the adenocarcinoma samples.
Figure 2
Figure 2
Correlations in relative expressions (2−ΔCt) of lncCDH5-3:3 and CDH1 (A) or EPCAM (B) genes in tumor samples from patients with NSCLC. Low and high expressions marked as “min” and “max”, respectively: for CDH1, the min > 0.13 and the max < 0.13; for EPCAM, the min > 1.6 and the max < 1.6; for IncCDH5-3:3, the min > 3.3 and the max > 3.3. The medians in these figures, (A,B), are marked as —, and the expression values of the lncCDH5-3:3, E-cadherin, or EpCAM patient samples are marked as red triangles and green or black circles, respectively.
Figure 3
Figure 3
Relative expressions (2−ΔCt) of lncCDH5-3:3 (A–C), EPCAM (D–F), and CDH1 (G–I), in A549, H1975, and H1703 cells, after lncCDH5-3:3 silencing. Expressions were evaluated at 24, 48, and 72 h post transient transfection, using RNA isolated from cultured cells. Data are presented as medians and minimum and maximum values. The p-values were calculated on the basis of the Friedman ANOVA and Dunn’s comparison post hoc tests. All statistically significant differences are marked: * p < 0.05 and ** p < 0.01. In this figure, the maximum values are marked as formula image; the minimum values are marked as formula image; the medians are represented by circles, squares, and triangles; formula image represents the p-value from the post hoc test.
Figure 4
Figure 4
The effects of lncCDH5-3:3 silencing on the living A549 or H1975 cells (percentage of cultured cells). The apoptosis estimation was carried out by Annexin V staining (A,B) and a caspase-3/7 activity assay (C,D). Analysis was performed at 24, 48, and 72 h post transient transfection. Data are presented as medians and minimum (formula image) and maximum (formula image) values from three biological replicates. The p-values were calculated on the basis of the Friedman ANOVA and Dunn’s comparative post hoc tests. Statistically significant results are marked: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. In this figure, the medians are represented as circles, squares, and triangles, and formula image represents the p-value from the post hoc test.
Figure 5
Figure 5
The effects of lncCDH5-3:3 silencing on the percentage of cultured cells of early apoptosis (A,B), and apoptotic (C–E) A549, H1975, and H1703 cells. The apoptosis estimation was carried out by Annexin V staining and a caspase-3/7 activity assay. Analysis was performed at 24, 48, and 72 h post transient transfection. Data are presented as medians and minimum (formula image) and maximum (formula image) values from three biological replicates. The p-values were calculated on the basis of the Friedman ANOVA and Dunn’s comparative post hoc tests. Statistically significant results are marked: ** p < 0.01, *** p < 0.005, and **** p < 0.0005. In this figure, the medians are represented by circles, squares, and triangles, and formula image represents the p-value from the post hoc test.
Figure 6
Figure 6
The effects of lncCDH5-3:3 silencing on the apoptosis intensity (cells in SubG1 gate) (A,B), and cell cycles (C–E) of cultured cells: A549, H1975, and H1703 cells. The cell cycle study was carried out by cell cycle assay. Analysis was performed at 24, 48, and 72 h post transient transfection. Data are presented as median values and minimum (formula image) and maximum (formula image) values from three biological replicates (n = 3). The p-values were calculated on the basis of the Friedman ANOVA and Dunn’s comparative post hoc tests. Statistically significant results are marked: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Medians are marked as circles, squares, and triangles; formula image represents the p-value from post hoc test.
Figure 7
Figure 7
The effects of lncCDH5-3:3 silencing on the distribution of cultured cells: A549 (A), H1975 (B), and H1703 cells (C) in the G2/M phase of the cell cycle. The cell cycle study was carried out by cell cycle assay. Analysis was performed at 24, 48, and 72 h post transient transfection. Data are presented as medians from three biological replicates. The p-values were calculated on the basis of the Friedman ANOVA and Dunn’s comparative post hoc tests. Statistically significant results are marked as * p < 0.05, ** p < 0.01, and **** p < 0.0005. In this figure, the maximum values are marked as formula image; the minimum values as formula image; the medians as circles, squares, and triangles; and formula image represents the p-value from the post hoc test.
Figure 8
Figure 8
The effects of lncCDH5-3:3 overexpression on the percentages of early apoptotic cells (A), or cells in the G0/G1 (B), S (C), and G2/M (D) cell cycle phases of the cultured cells of the A549, H1975, and H1703 cell lines. The effect of lncCDH5-3:3 silencing on the apoptosis intensity was carried out by Annexin V staining, while the cell cycle study was carried out by cell cycle assay. Data are presented as median values and minimum (formula image) and maximum (formula image) values from three biological replicates (n = 3). The p-values were calculated on the basis of the Wilcoxon test. Statistically significant results are marked as * p < 0.05 and ** p < 0.01; formula image represents the p-value from the post hoc test.
Figure 9
Figure 9
Western blot images (A), and fold changes (B) of Akt, pAkt, ERK1/2, and pERK1/2 expressions in response to lncCDH5-3:3 overexpression in cultured A549, H1975, and H1703 cells. In Western blot assay, β-actin was used as a loading control. The bar graph represents the median fold change and the minimum (formula image) and maximum (formula image) values of three independent replicates, and data are presented as fold changes relative to the control. The p-values were calculated on the basis of the Wilcoxon test. Statistically significant results are marked as ** p < 0.01; formula image represents the p-value from the post hoc test.
Figure 10
Figure 10
Western blot images (A), and fold changes (B) of pβ-catenin, Oct-4, EpCAM, E-cadherin, and Nanog expressions in response to lncCDH5-3:3 overexpression in cultured A549, H1975, and H1703 cells. β-actin was used as a loading control in the Western blot assay. The bar graph represents the median and the minimum (formula image) and maximum (formula image) values of three independent replicates, and the data are presented as fold changes relative to the control. The p-values were calculated on the basis of the Wilcoxon test. Statistically significant results are marked as * p < 0.05 and ** p < 0.01; formula image represents the p-value from the post hoc test.
Figure 11
Figure 11
The role of lncCDH5-3:3 in the cancer cells. The sharp arrowheads indicate the positive regulation of genes by lncCDH5-3:3, while the flat heads indicate the negative regulation, which causes the degradation of β-catenin. The crossed flathead arrows show the lack of protein interactions reported in the literature.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Zappa C., Mousa S.A. Non-small cell lung cancer: Current treatment and future advances. Transl. Lung Cancer Res. 2016;5:288–300. doi: 10.21037/tlcr.2016.06.07. - DOI - PMC - PubMed
    1. Chen S., Shen X. Long noncoding RNAs: Functions and mechanisms in colon cancer. Mol. Cancer. 2020;19:167. doi: 10.1186/s12943-020-01287-2. - DOI - PMC - PubMed
    1. Ward M., McEwan C., Mills J.D., Janitz M. Conservation and tissue-specific transcription patterns of long noncoding RNAs. J. Hum. Transcr. 2015;1:2–9. doi: 10.3109/23324015.2015.1077591. - DOI - PMC - PubMed
    1. Nie L., Wu H.-J., Hsu J.-M., Chang L.S.-S., Labaff A.M., Li C.-W., Wang Y., Hsu J.L., Hung M.-C. Long non-coding RNAs: Versatile master regulators of gene expression and crucial players in cancer. Am. J. Transl. Res. 2012;4:127–150. - PMC - PubMed
    1. Zhang X., Wang W., Zhu W., Dong J., Cheng Y., Yin Z., Shen F. Mechanisms and Functions of Long Non-Coding RNAs at Multiple Regulatory Levels. Int. J. Mol. Sci. 2019;20:5573. doi: 10.3390/ijms20225573. - DOI - PMC - PubMed

Publication types