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. 2019 Sep;55(3):708-720.
doi: 10.3892/ijo.2019.4848. Epub 2019 Jul 25.

"V体育官网入口" Linc00961 inhibits the proliferation and invasion of skin melanoma by targeting the miR‑367/PTEN axis

Xin Mu  1 , Kuan-Hou Mou  1 , Rui Ge  1 , Dan Han  1 , Yan Zhou  1 , Li-Juan Wang  1
Affiliations

Linc00961 inhibits the proliferation and invasion of skin melanoma by targeting the miR‑367/PTEN axis

Xin Mu et al. Int J Oncol. 2019 Sep.

Erratum in

Abstract

Long intergenic noncoding RNA 00961 (Linc00961) has been identified as a tumor suppressor in various types of cancer. However, the critical roles of Linc00961 in the carcinogenesis and progression of skin melanoma (SM) are yet to be fully elucidated. The present study revealed via reverse transcription‑quantitative PCR analysis that Linc00961 was downregulated in the tissues of patients with SM compared with benign nevi, and in A375, A2058 and SK‑MEL‑28 cell lines compared with human melanocytes. Furthermore, overexpression of Linc00961 inhibited cell proliferation, and promoted the apoptosis of A375 and SK‑MEL‑28 cells in vitro and in vivo, as determined by Cell Counting Kit‑8 and flow cytometry assays, and tumor xenograft studies, respectively. Overexpression of Linc00961 also led to an attenuation of the migration and invasive capabilities of A375 and SK‑MEL‑28 cells, measured using Transwell assays. Functionally, it was demonstrated that Linc00961 acted as a competing endogenous RNA (ceRNA) by competitively sponging microRNA‑367 (miR‑367) in A375 and SK‑MEL‑28 cells; restoration of miR‑367 rescued the inhibitory effects of Linc00961 on A375 and SK‑MEL‑28 cells. Finally, it was observed that phosphate and tension homology deleted on chromosome 10 (PTEN), an established target of miR‑367 in A375 and SK‑MEL‑28 cells, was positively regulated by Linc00961, and its inhibition reversed the inhibitory effects of Linc00961 on the proliferation and invasion of A375 and SK‑MEL‑28 cells VSports手机版. Collectively, the present study revealed that Linc00961 was downregulated in SM, and furthermore, Linc00961 was identified as a ceRNA that inhibits the proliferation and invasion of A375 and SK‑MEL‑28 cells by modulating the miR‑367/PTEN axis. .

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Figures

Figure 1
Figure 1
Linc00961 is downregulated in SM cells and the tissues of patients with SM. (A) Expression of Linc00961 in tissues of SM and BN. (B) Patients with SM in GSM3071633 exhibited decreased expression of Linc00961 compared with patients with BN. (C) Relative expression levels of Linc00961 in human epidermal melanocytes and SM cells. (D) Association of Linc00961 expression with the overall survival of SM patients. The Cancer Genome Atlas data were analyzed using the Gene Expression Profiling Interactive Analysis website. Data from three experiments are presented as mean ± SD. *P<0.05 vs. BN or HEMa-LP. Linc00961, long intergenic noncoding RNA 00961; SM, skin melanoma; BN, benign nevi; HR, hazard ratio.
Figure 2
Figure 2
Effects of Linc00961 on the proliferation and apoptosis of A375 and SK-MEL-28 cells. Effects of Linc00961 on the proliferation of (A) A375 and (B) SK-MEL-28 cells, as determined using a Cell Counting Kit-8 assay. (C) Effects of Linc00961 on the apoptosis of A375 and SK-MEL-28 cells, detected via flow cytometry. (D) Statistical analysis of the early apoptotic rates of A375 and SK-MEL-28 cells infected with Lv-control and Lv-Linc00961. (E) Tumor growth curve of A375 cells transfected with Lv-control and Lv-Linc00961. (F) Digital pictures of the resected tumors of animals injected with A375 cells infected with Lv-control (n=3) and Lv-Linc00961 (n=3), captured after 35 days. The maximum diameters of Lv-control and Lv-Linc00961 were 1.718 and 0.737 cm, respectively. Scale bar=1 cm. Data from three experiments are presented as the mean ± SD. *P<0.05 vs. Lv-control. Linc00961, long intergenic noncoding RNA 00961; Lv, lentivirus; PI, propidium iodide.
Figure 3
Figure 3
Effects of Linc00961 on proliferation and apoptosis in tumors from nude mice injected with A375 cells. (A) Ki-67 positive cells in nude mice bearing A375 cells transfected with Lv-control and Lv-Linc00961. (B) Statistical analysis of the percentage of Ki-67 positive cells in xenograft tumors. (C) TUNEL-positive cells in nude mice bearing A375 cells transfected with Lv-control and Lv-Linc00961. (D) Statistical analysis of the percentage of TUNEL-positive cells in xenograft tumors. Scale bar=50 µm. Data from three experiments are presented as the mean ± SD. *P<0.05 vs. Lv-control. Linc00961, long intergenic noncoding RNA 00961; Lv, lentivirus.
Figure 4
Figure 4
Effects of Linc00961 on the migration and invasion of SM cells. (A) Effects of Linc00961 on the migration of A375 and SK-MEL-28 cells, as determined by a Transwell assay. (B) Statistical analysis of the numbers of migratory A375 and SK-MEL-228 cells. (C) Effects of Linc00961 on cell invasion of A375 and SK-MEL-28 cells, as determined by a Matrigel-coated Transwell assay. (D) Statistical analysis of the numbers of invasive A375 and SK-MEL-228 cells. Scale bar=20 µm. Data from three experiments are presented as the mean ± SD. *P<0.05 vs. Lv-control. LINC00961, long intergenic noncoding RNA 00961; Lv, lentivirus.
Figure 5
Figure 5
Linc00961 expression is inversely correlated with miR-367 expression in SM. Effects of Linc00961 on the expression of miRNAs that possessed putative binding sites for Linc00961 in (A) A375 and (B) SK-MEL-28 cells. (C) Expression levels of miR-367 in SM and BN tissues. (D) Association between miR-367 and Linc00961 in SM tissues. (E) Expression levels of miR-367 in SM cells and human epidermal melanocytes. (F) WT and MUT targeting regions of Linc00961 for miR-367. Data from three experiments are presented as the mean ± SD. *P<0.05 vs. control (Lv-control, BN, HEMa-LP). Linc00961, long intergenic noncoding RNA 00961; miR, microRNA; SM, skin melanoma; BN, benign nevi; WT, wild-type; MUT, mutant.
Figure 6
Figure 6
Linc00961 directly sponges miR-367 in SM cells. (A) Effects of Linc00961 on the expression of miR-367 in SM cells. Relative luciferase activity in (B) A375 and (C) SK-MEL-28 cells co-transfected with Linc00961-WT or LINC00961-MUT luciferase plasmid and miR-367 NC or mimics. (D) Detection of LINC00961 in RNA pulled down by Bio-Linc00961, Bio-Linc00961-MUT or Bio-NC probes. Detection of miR-367 in RNA pulled down by Bio-Linc00961, Bio-Linc00961-MUT or Bio-NC probes in (E) A375 and (F) SK-MEL-28 cells. Data from three experiments are presented as the mean ± SD. *P<0.05 vs. control (Lv-control, miR-367 NC, Bio-NC-probe). Linc00961, long intergenic noncoding RNA 00961; miR, microRNA; SM, skin melanoma; NC, negative control; WT, wild-type; MUT, mutant; Bio, biotinylated.
Figure 7
Figure 7
Upregulation of miR-367 reverses the effects of Linc00961 on the proliferation and apoptosis of skin melanoma cells. (A) Expression of miR-367 in A375 and SK-MEL-28 cells transfected with Lv-control + miR-367 NC, Lv-Linc00961 + miR-367 NC or Lv-Linc00961 + miR-367 mimics, as determined via reverse transcription-quantitative PCR. Proliferation of (B) A375 and (C) SK-MEL-28 cells transfected with Lv-control + miR-367 NC, Lv-Linc00961 + miR-367 NC or Lv-Linc00961 + miR-367 mimics, as determined by a Cell Counting Kit-8 assay. (D) Apoptosis of A375 and SK-MEL-28 cells transfected with Lv-control + miR-367 NC, Lv-Linc00961 + miR-367 NC or Lv-Linc00961 + miR-367 mimics, as determined via flow cytometry. (E) Statistical analysis of early apoptotic rates of A375 and SK-MEL-228 cells transfected with Lv-control + miR-367 NC, Lv-Linc00961 + miR-367 NC or Lv-Linc00961 + miR-367 mimics. Data from three experiments are presented as the mean ± SD. *P<0.05 vs. Lv-control + miR-367 NC. Linc00961, long intergenic noncoding RNA 00961; Lv, lentivirus; miR, microRNA; NC, negative control; PI, propidium iodide.
Figure 8
Figure 8
Upregulation of miR-367 reverses the effects of Linc00961 on the migration and invasion of SM cells. (A) Numbers of migratory A375 and SK-MEL-28 cells transfected with Lv-control + miR-367 NC, Lv-Linc00961 + miR-367 NC or Lv-Linc00961 + miR-367 mimics, determined by a Transwell assay. (B) Statistical analysis of the numbers of migratory A375 and SK-MEL-228 cells. (C) Numbers of invasive A375 and SK-MEL-28 cells transfected with Lv-control + miR-367 NC, Lv-Linc00961 + miR-367 NC or Lv-Linc00961 + miR-367 mimics, as determined by a Transwell assay. (D) Statistical analysis of the numbers of invasive A375 and SK-MEL-228 cells. Scale bar=20 µm. Data from three experiments are presented as the mean ± SD. *P<0.05 vs. Lv-control + miR-367 NC. Linc00961, long intergenic noncoding RNA 00961; Lv, lentivirus; miR, microRNA; NC, negative control.
Figure 9
Figure 9
Silencing PTEN reverses the inhibitory effects of Linc00961 on the proliferation and invasion of SM cells. (A) mRNA and (B) protein expression of PTEN in A375 and SK MEL-28 cells transfected with Lv-control + miR-367 NC, Lv-Linc00961 + miR-367 NC or Lv-Linc00961 + miR-367 mimics. Proliferation of (C) A375 and (D) SK-MEL-28 cells transfected with Lv-control + siRNA NC, Lv-Linc00961 + siRNA NC or Lv-Linc00961 + PTEN siRNA, as determined by a Cell Counting Kit-8 assay. (E) Apoptosis of A375 and SK-MEL-28 cells transfected with Lv-control + siRNA NC, Lv-Linc00961 + siRNA NC or Lv-Linc00961 + PTEN siRNA, as determined via flow cytometry. Numbers of (F) migratory and (G) invasive A375 and SK-MEL-28 cells transfected with Lv-control + siRNA NC, Lv-Linc00961 + siRNA NC, or Lv-Linc00961 + PTEN siRNA, as determined via Transwell assays. Data from three experiments are presented as mean ± SD. *P<0.05 vs. Lv-control + miR-367 NC or siRNA NC. Linc00961, long intergenic noncoding RNA 00961; PTEN, phosphate and tension homology deleted on chromosome 10; Lv, lentivirus; miR, microRNA; siRNA, small interfering RNA; NC, negative control.
Figure 10
Figure 10
Mechanism underlying the regulatory functions of Linc00961 in skin melanoma. Linc00961 inhibits the proliferation and invasion of SM cells by targeting the miR-367/PTEN axis. Linc00961, long intergenic noncoding RNA 00961; miR-367, microRNA-367; PTEN, phosphate and tension homology deleted on chromosome 10; RISC, RNA-induced silencing complex.
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