The long noncoding RNA AK002107 negatively modulates miR-140-5p and targets TGFBR1 to induce epithelial-mesenchymal transition in hepatocellular carcinoma

Abstract

The abnormal expression of long noncoding RNAs (lncRNAs) is associated with human carcinoma. The present study aimed to investigate the mechanisms underlying the function of lncRNA AK002107 in the progression of hepatocellular carcinoma (HCC). The differential expression of lncRNAs between HCC and paired nontumor tissues was identified using microarrays, and the correlation between the expression of lncRNA AK002107 and the clinical prognosis of HCC was analyzed. We investigated the role of lncRNA AK002107 in HCC tumor biology in vitro using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), colony formation, and Matrigel invasion assays and in vivo by assessing the growth of xenografted HCC tumors. The potential microRNAs that interact with lncRNA AK002107 were identified using online tools and were verified using PCR and luciferase reporter assay. The levels of TGFBR1, E-cadherin, and vimentin were determined using western blot assays. We then further investigated the correlation between expression of lncRNA AK002107 with miR-140-5p and TGFBR1 expression in HCC tissues. The expression of lncRNA AK002107 is frequently upregulated in HCC samples and cell lines. Patients with HCC who have elevated lncRNA AK002107 expression exhibit poorer overall survival and disease-free survival. Silencing lncRNA AK002107 expression significantly inhibited HCC cell proliferation, colony formation, and invasion both in vitro and in vivo. Furthermore, lncRNA AK002107 directly binds to miR-140-5p and significantly inhibits miR-140-5p expression. The functions of lncRNA AK002107 in cell growth and tumor invasion are mediated via miR-140-5p. lncRNA AK002107 upregulated TGFBR1 expression and then induced epithelial-mesenchymal transition (EMT) by inhibiting miR-140-5p expression. The expression of lncRNA AK002107 inversely correlated with miR-140-5p expression and positively correlated with TGFBR1 expression in HCC tissues. In summary, lncRNA AK002107 functions as an oncogene in tumors by inhibiting miR-140-5p, targeting TGFBR1, and then inducing EMT. The lncRNA AK002107/miR-140-5p/TGFBR1/EMT regulatory network may be a valuable target for the development of novel diagnostic and treatment methods for HCC.

Keywords: epithelial-mesenchymal transition; hepatocellular carcinoma; lncRNA AK002107; miR-140-5p.

Figure 1

Overexpression of lncRNA AK002107 in HCC tissues. (A) Hierarchical clustering analysis of the differentially expressed lncRNAs (> 2‐fold; P < 0.05) between HCC samples (T, tumor) and paired nontumor samples (N, nontumor). (B) The expression of lncRNA AK002107 in thirty fresh HCC samples and adjacent noncancerous tissues was analyzed using qRT‐PCR. Results expressed as the means ± SDs. Student's t‐test was performed for statistical comparisons. (C) Representative images of high and low lncRNA AK002107 expression in 100 human HCC tissues analyzed using ISH (200× and 400×). Positive ISH staining for lncRNA AK002107 was blue (arrow). The length of the scale bars is 100 μm (200×) and 50 μm (400×), respectively. (D) Kaplan–Meier survival curves showing the overall survival of 100 patients with HCC classified according to their relative lncRNA AK002107 expression level; P < 0.001 according to the log‐rank test. (E) Kaplan–Meier survival curves showing the disease‐free survival of 100 patients with HCC classified according to their relative lncRNA AK002107 expression level; P < 0.001 according to the log‐rank test.

Figure 2

lncRNA AK002107 promotes HCC proliferation, colony formation, and invasion in vitro. (A) The expression of lncRNA AK002107 in six human HCC cell lines, including MHCC97H, MHCC97L, BEL7402, SMMC7721, Hep3B, and HepG2, was upregulated compared to the normal cell line LO₂, as determined using qRT‐PCR. *P < 0.05 and **P < 0.01. (B) The expression of lncRNA AK002107 was detected in BEL7402 and MHCC97H cells using qRT‐PCR after the transduction of lentiviruses encoding lncRNA AK002107 short hairpin RNA (shRNA)‐1, shRNA‐2, or scrambled shRNA. All qPCR results shown as data from at least two independent replicates. Statistical analyses using one‐way ANOVA with Tukey's post‐test, *P < 0.05. and **P < 0.01. (C) MTT assays were performed to assess the proliferation of the HCC cell lines BEL7402 and MHCC97H in response to transfection with lncRNA AK002107 shRNA‐1 or lncRNA AK002107 shRNA‐2 compared with the sh‐NC group. Two‐way ANOVA with multiple comparisons (Tukey's post t‐test) performed to measure significance, *P < 0.05. Results are from three independent experiments. (D) A colony formation assay was performed to determine the colony formation capacity of the HCC cell lines BEL7402 and MHCC97H in response to transfection with lncRNA AK002107 shRNA‐1 or lncRNA AK002107 shRNA‐2 compared with the sh‐NC group. Magnification, 200×. Error bars represent the SD of three independent experiments. Student's t‐test was used to statistical analyses. **P < 0.01. (E) Transwell invasion assay of BEL7402 and MHCC97H cells transfected with lncRNA AK002107 shRNA‐1 or lncRNA AK002107 shRNA‐2 compared with the sh‐NC group. The length of the scale bars is 100 μm. Error bars represent the SD of three independent experiments. Student's t‐test was used to statistical analyses. **P < 0.01.

Figure 3

Downregulation of lncRNA AK002107‐1 expression inhibits HCC tumor growth in vivo. (A) Representative images of tumors formed in nude mice that had been subcutaneously injected with BEL7402 or MHCC97H cells transfected with lncRNA‐AK002107 shRNA‐1 compared with the sh‐NC group. (B) The weight of tumors dissected from the shAK002107‐1 group was lower than the sh‐NC group. Data are shown as means ± SD, and Student's t‐test was used to calculate significance, *P < 0 .05. (C) The tumor volumes were significantly reduced at 1, 2, 3, and 4 weeks following the injection of shAK002107‐1‐expressing tumor cells compared with the sh‐NC group. Data are shown as means ± SD, and Student's t‐test was used to calculate significance,*P < 0.05. (D) Representative ISH image showing lncRNA AK002107 shRNA‐1 expression in xenograft tumors from nude mice injected subcutaneously with BEL7402 or MHCC97H cells transfected with lncRNA AK002107 shRNA‐1 construct compared with the sh‐NC group. The length of the scale bars is 50 μm. (E) Levels of the E‐cadherin and vimentin proteins in xenograft tissues were determined using western blotting.

Figure 4

MiR‐140‐5p is a target of lncRNA AK002107. (A) Bioinformatics analysis identified the putative binding sites for lncRNA AK002107 in the miR‐140‐5p sequence. (B) Luciferase activity was determined in 293T cells cotransfected with miRNAs (control mimics or miR‐140‐5p mimics) and a reporter vector containing lncRNA AK002107 segments (WT or MUT) that bind to miR‐140‐5p. *P < 0.05. (C) The expression of miR‐140‐5p in six human HCC cell lines, including MHCC97H, MHCC97L, BEL7402, SMMC7721, Hep3B, and HepG2, was downregulated compared to the normal cell line LO₂, as determined using qRT‐PCR. *P < 0.05 and **P < 0.01. (D) The expression of miR‐140‐5p was detected in BEL7402 and MHCC97H cells using qRT‐PCR after the transduction of lentiviruses encoding lncRNA AK002107 short hairpin RNA (shRNA)‐1, shRNA‐2, or scrambled shRNA. All qPCR results shown as data from at least two independent replicates. Statistical analyses using one‐way ANOVA with Tukey's post‐test, *P < 0.05 and **P < 0.01.

Figure 5

lncRNA AK002107 plays a role in HCC cell proliferation and invasion by negatively regulating miR‐140‐5p expression. (A) MTT assays were performed to assess the proliferation of the HCC cell lines BEL7402 and MHCC97H in response to the transfection of lncRNA AK002107 shRNA‐1, lncRNA AK002107 shRNA‐2, or miR‐140‐5p inhibitors or cotransfection of sh‐lncRNA‐AK002107‐1 or sh‐lncRNA‐AK002107‐2 with miR‐140‐5p inhibitors compared with the sh‐NC group. Two‐way ANOVA with multiple comparisons (Tukey's post t‐test) performed to measure significance, *P < 0.05. Results are from three independent experiments. (B) Transwell invasion assay of BEL7402 and MHCC97H cells transfected with lncRNA AK002107 shRNA‐1, lncRNA AK002107 shRNA‐2, or miR‐140‐5p inhibitor or cotransfection of sh‐lncRNA‐AK002107‐1 or sh‐lncRNA‐AK002107‐2 with miR‐140‐5p inhibitors compared with the sh‐NC group. The length of the scale bars is 100 μm. Error bars represent the SD of three independent experiments. Student's t‐test was used to statistical analyses. *P < 0.05 and **P < 0.01.

Figure 6

lncRNA AK002107 modulates the expression of miR‐140‐5p, targets TGFBR1, and induces EMT. (A) The putative binding sites between TGFBR1 and miR‐140‐5p. (B) Luciferase activity was determined in 293T cells cotransfected with miRNAs (control mimics or miR‐140‐5p mimics) and a reporter vector containing TGFBR1 3′‐UTR segments (WT or MUT) that bind to miR‐140‐5p. Error bars represent the SD of three independent experiments. Student's t‐test was used to statistical analyses. *P < 0.05 (C) TGFBR1 expression was detected in BEL7402 and MHCC97H cells using qRT‐PCR after the transduction of lentiviruses encoding lncRNA AK002107 short hairpin RNA (shRNA)‐1, shRNA‐2, or miR‐140‐5p inhibitor, cotransfection of the miR‐140‐5p inhibitor and sh‐lncRNA‐AK002107‐1, and cotransfection of the miR‐140‐5p inhibitor and sh‐lncRNA‐AK002107‐2 or scrambled shRNA. Error bars represent the SD of three independent experiments. Student's t‐test was used to statistical analyses. *P < 0.05 and **P < 0.01. (D) Levels of the E‐cadherin, vimentin, and TGFBR1 proteins were determined in BEL7402 and MHCC97H cells transfected with lentiviruses encoding sh‐lncRNA‐AK002107‐1, sh‐lncRNA‐AK002107‐2, or miR‐140‐5p inhibitor, cotransfection of the miR‐140‐5p inhibitor and sh‐lncRNA‐AK002107‐1, or cotransfection of the miR‐140‐5p inhibitor and sh‐lncRNA‐AK002107‐2 or scrambled shRNA using western blot assays.

Figure 7

Expression of miR‐140‐5p and TGFBR1 and the correlations between lncRNA AK002107 and miR‐140‐5p and TGFBR1 expression in HCC tissues. (A) The expression of miR‐140‐5p was analyzed in thirty fresh HCC samples and adjacent noncancerous tissues using qRT‐PCR. Results expressed as the means ± SDs. Student's t‐test was performed for statistical comparisons. P < 0.0001, (B) Correlations between the expression of lncRNA AK002107 and miR‐140‐5p were analyzed using Spearman's rank correlation analysis. (C) TGFBR1 expression in thirty fresh HCC samples and adjacent noncancerous tissues was analyzed using qRT‐PCR. Results expressed as the means ± SDs. Student's t‐test was performed for statistical comparisons. P = 0.006. (D) Correlations between the expression of lncRNA AK002107 and TGFBR1 were analyzed using Spearman's rank correlation analysis. (E) Schematic of the lncRNA AK002107/miR‐140‐5p/TGFBR1/EMT regulatory network in HCC.