MicroRNA-30e-5p promotes cell growth by targeting PTPN13 and indicates poor survival and recurrence in lung adenocarcinoma

Abstract

Aberrant microRNA expression is involved in the regulation of various cellular processes, such as proliferation and metastasis in multiple diseases including cancers. MicroRNA-30e-5p (miR-30e) was previously reported as an oncogenic or tumour suppressing miRNA in some malignancies, but its function in lung adenocarcinoma (LAC) remains largely undefined. In this study, we found that the expression of miR-30e was increased in LAC tissues and cell lines, associated with tumour size and represented an independent prognostic factor for overall survival and recurrence of LAC patients. Further functional experiments showed that knockdown of miR-30e suppressed cell growth while its overexpression promoted growth of LAC cells and xenografts in vitro and in vivo. Mechanistically, PTPN13 was identified as the direct target of miR-30e in LAC, in which PTPN13 expression was down-regulated in LAC tissues and showed the inverse correlation with miR-30e expression. Overexpression of PTPN13 inhibited cell growth and rescued the proliferation-promoting effect of miR-30e through inhibition of the EGFR signalling. Altogether, our findings suggest that miR-30e could function as an oncogene in LAC via targeting PTPN13 and act as a potential therapeutic target for treating LAC.

Keywords: PTPN13; growth; lung adenocarcinoma; microRNA-30e-5p.

Figure 1

The expression of miR‐30e was up‐regulated in LAC. (A) miR‐30e had the differential expression in 452 LAC patient samples and 46 adjacent normal tissues indicated by a 2015 TCGA data set. (B) TCGA showed that miR‐30e expression was increased in LAC compared to the adjacent normal tissues. The ordinate value reflected the relative ratio of miR‐30e expression in LAC tissue versus adjacent normal tissue. (C) miR‐30e expression was significantly up‐regulated in 67.95% (53/78) of LAC patient tissues compared to the adjacent normal. (D) The expression level of miR‐30e in LAC patients with tumour size≥3 cm and tumour size<3 cm compared with the adjacent non‐tumour tissues (ANTT). (E, G) Overall survival and (F, H) recurrence curves demonstrated the correlation of miR‐30e high expression or low expression with the percent survival and recurrence in LAC patients.

Figure 2

Knockdown of miR‐30e inhibited cell proliferation and colony formation. (A) The expression levels of miR‐30e in different LAC cell lines. (B) The expression level of miR‐30e was measured by real‐time PCR. (C) The effects of miR‐30e knockdown on cell proliferation by MTT assay. (D) The effects of miR‐30e knockdown on cell colony formation. (E) The effects of miR‐30e knockdown on cell cycle distribution. *P < 0.05, **P < 0.01.

Figure 3

PTPN13 was identified as a direct target of miR‐30e. (A) The main target genes of miR‐30e in cancer tissues. (B) Diagrams showed the miR‐30e putative binding sites and corresponding mutant sites of PTPN13. (C) The expression level of PTPN13 was examined after transfection with miR‐30e by real‐time PCR and Western blotting assays. (D) Luciferase activity was detected after miR‐30e transfection. (E) The expression level of PTPN13 was examined after transfection with sh‐miR‐30e by real‐time PCR and Western blotting assays. *P < 0.05, **P < 0.01.

Figure 4

Enforced expression of PTPN13 inhibited cell proliferation and colony formation. (A) TCGA showed a decreased expression of PTPN13 in LAC compared to the adjacent normal tissues, and a negative correlation of PTPN13 with miR‐30E expression in LAC. The expression levels of PTPN13 were determined in human LAC tissues (B) and cells (C) by real‐time PCR and Western blot assays. (D) The effects of PTPN13 overexpression on cell proliferation by MTT assay. (E) The effects of PTPN13 overexpression on cell colony formation. (F) The effect of sh‐PTPN13 vector transfection for 48 hrs on PTPN13 mRNA and protein expression. (G) The effects of PTPN13 depletion on cell proliferation by MTT assay. **P < 0.01.

Figure 5

PTPN13 rescued tumour proliferation‐promoting effects by miR‐30e. (A and B) The effects of PTPN13 overexpression on cell proliferation in miR‐30e‐transfected A549 and NCI‐H23 cells indicated by MTT assay. (C and D) The effects of PTPN13 overexpression on the protein expression of EGFR/AKT pathway in miR‐30e‐transfected A549 and NCI‐H23 cells indicated by western blotting. (E and F) The effects of PTPN13 knockdown on the protein expression of EGFR/AKT pathway in sh‐miR‐30e‐transfected A549 and NCI‐H23 cells indicated by Western blotting.

Figure 6

miR‐30e overexpression inhibited A549 xenograft tumour growth. (A) The effects of miR‐30e overexpression on proliferation activity of A549 xenograft tumours. (B and C) The effects of miR‐30e overexpression on the average weight and volumes in xenograft tumours. (D) The expression level of miR‐30e and PTPN13 in LAC tissues from miR‐NC and miR‐30e groups. **P < 0.01.