Long noncoding RNA PP7080 promotes hepatocellular carcinoma development by sponging mir-601 and targeting SIRT1

Figures & data

Figure 1. LncRNA PP7080 is highly expressed in HCC and suggests a poor survival prognosis

(a) Analysis of the expression of lncRNA PP7080 in liver cancer based on the transcriptome data of HCC cancer tissue (369) and normal tissue (50) in the TCGA database. (b) The expression level of lncRNA PP7080 mRNA detected by RT-PCR in 40 pairs of HCC cancer tissues and adjacent tissues. (c) Survival curve drawn by KM-plotter on the expression of lncRNA PP7080 in 40 patients. (d) The expression level of lncRNA PP7080 mRNA detected by RT-PCR in HCC cell lines (SMMC7721, Huh7, HepG2, HCCLM3 and SK-HEP-1) and the normal liver cell lines (L02). Data were representative of three independent experiments and expressed as mean ± SD (*p < 0.05, **p < 0.01).

Table 1. PP7080 expression and clinical characteristics of the HCC patients

Variable	Number	PP7080 expression		p-Value
		Low	High
Age (years)				0.68
<60	11	7	4
≥60	29	15	14
Gender				0.93
Male	22	14	8
Female	18	10	8
Viral hepatitis				0.95
Type B	37	22	15
Type B and C	3	2	1
Tumor size (cm)				0.03
<3	10	7	3
≥3	30	6	24
TNM				0.02
I–II	19	15	4
III–IV	21	8	13
Vascular invasion				0.02
No	23	17	6
Yes	17	6	11
Lymph nodes metastasis				0.67
No	22	16	6
Yes	18	6	12
Serum AFP (ng/ml)				0.93
<25	11	6	5
≥25	29	17	12

Figure 2. Knockdown of LncRNA PP7080 inhibits the proliferation, migration and invasion of HCC cells

(a) Detection of knockdown efficiency of lncRNA PP7080 by qRT-PCR. (b) Knockdown of lncRNA PP7080 induced HepG2 and Huh7 cell viability detection by CCK8 assay of sh-NC group and sh-PP7080 group. (c) Knockdown of lncRNA PP7080 induced HepG2 colony forming ability detection by colony forming assay of sh-NC group and Sh-PP7080 group. (d) The effect of knockdown of lncRNA PP7080 on HepG2 and Huh7 cell migration detected by transwell experiment. (e) The effect of knockdown of lncRNA PP7080 on HepG2 and Huh7 cell invasion detected by transwell experiment (with Matrigel). Data are representative of three independent experiments and expressed as mean ± SD (**p < 0.01).

Figure 3. LncRNA PP7080 regulates HCC by targeting miR-601

(a) The targeted binding site of lncRNA PP7080 is predicted online by DIANA TOOLS software, the binding site is displayed and the mutation sequence of the site is designed. (b) The above-mentioned binding site and the mutated sequence were constructed into the fluorescein reporter gene, and the expression of fluorescein was detected after HepG2 or Huh7 was transfected with miR-NC or miR-601, respectively. (c) The effect of knockdown of lncRNA PP7080 on the expression of miR-601 was detected by qRT-PCR. (d) The expression levels of miR-601 in 40 pairs of HCC cancer tissues and adjacent tissues were detected by qRT-PCR. (e) The Pearson correlation analysis of the expression of lncRNA PP7080 and miR-601 in the cancer tissues of 40 cancer patients. Data are representative of three independent experiments and expressed as mean ± SD (***p < 0.001).

Figure 4. MiR-601 targets and regulates the expression of SIRT1 gene in HCC

(a) The sequence region of miR-601 targeted to bind SIRT1 was predicted online by Starbase software. The figure shows the binding site and design of the mutation sequence of the site. (b) The above-mentioned binding site and the mutated sequence were constructed into the fluorescein reporter gene, and the expression of fluorescein was detected after HepG2 or Huh7 was transfected with miR-NC or miR-601, respectively. (c) The expression levels of SIRT1 in 40 pairs of HCC cancer tissues and adjacent tissues were detected by qRT-PCR. (d, e) The Pearson correlation analysis of the expression of SIRT1 and lncRNA PP7080 or miR-601 in the cancer tissues of 40 cancer patients. (f, g) The effect of knockdown of lncRNA PP7080 or miR-601 on the expression of SIRT1 was detected by qRT-PCR in HepG2 or Huh7 cells. (g–i) The effect of knockdown of lncRNA PP7080 or miR-601 on the expression of SIRT1 was detected by western blot in HepG2 or Huh7 cells. Data are representative of three independent experiments and expressed as mean ± SD (**p < 0.01; ***p < 0.001).

Figure 5. LncRNA PP7080 promotes the malignant progression of HCC through the miR-601/SIRT1 signal axis

(a) The inhibition efficiency of miR-601inh on mir-601 was detected by qRT-PCR. (b) Transfection efficiency test of SIRT1 overexpression in HepG2 or Huh7, the expression level of SIRT1 mRNA was detected by qRT-PCR. (c, d) Detection of the expression of SIRT1 protein in HepG2 or Huh7 after cells were co-transfected with miR-601inh or OE-SIRT1 after lncRNA PP7080 knockdown by WB of sh-NC group, sh-PP7080#1 group, sh-PP7080#1+ miR-601inh group and sh-PP7080#1+ OE-SIRT1 group. (e, f) Co-transfected with miR-601inh or OE-SIRT1 after lncRNA PP7080 knockdown induced HepG2 or Huh7 cell viability detection by CCK8 assay of sh-NC group, sh-PP7080#1 group, sh-PP7080#1+ miR-601inh group and sh-PP7080#1+ OE-SIRT1 group. (g) The effect of co-transfected with miR-601inh or OE-SIRT1 after lncRNA PP7080 knockdown on Huh7 and HepG2 cell colony forming detected by transwell experiment. (h) The effect of co-transfected with miR-601inh or OE-SIRT1 after lncRNA PP7080 knockdown on Huh7 and HepG2 cell migration detected by transwell experiment. (i) The effect of co-transfected with miR-601inh or OE-SIRT1 after lncRNA PP7080 knockdown on Huh7 and HepG2 cell invasion detected by transwell experiment (with Matrigel). Data are representative of three independent experiments and expressed as mean ± SD (**p < 0.01; ***p< 0.001).