LINC00339 promotes growth and invasiveness of hepatocellular carcinoma by the miR-1182/SKA1 pathway

Abstract

Background: Extensive research has shown that long noncoding RNA (lncRNA) is involved in tumorigenesis, including hepatocellular carcinoma (HCC). The lncRNA LINC00339 was reported to regulate the development of lung cancer or breast cancer. However, whether LINC00339 participates in HCC progression remains unclear. Here, our results showed that LINC00339 was upregulated in HCC. Methods: qRT-PCR and in situ hybridization (ISH) was used to analyze LINC00339 expression in tumor tissues and cell lines. CCK8 and colony formation assays were used to analyze cell proliferation. Transwell assay was used to analyze cell migration and invasion. Xenograft experiment was used to test tumor growth in vivo. Results: LINC00339 overexpression was correlated with an advanced stage, metastasis, and bad prognosis in HCC patients. Functional investigation showed that LINC00339 knockdown significantly suppressed HCC cell proliferation, migration, and invasion. Moreover, decreased LINC00339 expression inhibited HCC growth in vivo. Mechanistically, LINC00339 could interact with miR-1182 to promote SKA1 expression. We also demonstrated that SKA1 acted as an oncogene and SKA1 upregulation reversed the effect of LINC00339 silencing. Conclusion: Our results illustrated that the LINC00339/miR-1182/SKA1 axis plays an essential role in HCC progression.

Keywords: LINC00339; SKA1; hepatocellular carcinoma; miR-1182; progression.

Figure 1

LINC00339 expression was elevated in HCC. (A) The expression of LINC00339 in HCC tissues and normal tissues was measured by qRT-PCR. (B) The expression of LINC00339 in HCC tissues of stage I/II and stage III/IV was analyzed by qRT-PCR. (C) In situ hybridization assay showed that LINC00339 expression was positively correlated with metastasis in HCC. (D) qRT-PCR analysis indicated that LINC00339 expression was increased in metastatic HCC tissues. (E) Relative expression of LINC00339 in HCC cell lines and LO2 cells. (F) Increased expression of LINC00339 in HCC patients predicted a low survival rate. **p<0.01 and ***p<0.001. All experiments were repeated three times. Abbreviations: HCC, hepatocellular carcinoma; qRT-PCR, quantitative reverse transcription PCR.

Figure 2

LINC00339 knockdown led to attenuated proliferation, migration, and invasion of HCC cells. (A) Relative expression of LINC00339 in SMMC-7721 and Hep3B cells transfected with indicated plasmids. (B and C) CCK8 and EdU assays showed that LINC00339 knockdown inhibited the proliferation of HCC cells. (D) Colony formation assay indicated that the colony number was decreased after LINC00339 knockdown. (E and F) Transwell assay showed that the abilities of migration and invasion were weakened by LINC00339 knockdown. (G) Tumor volumes were measured. (H) Tumor weights were determined after 5 weeks. *p<0.05. All experiments were repeated three times. Abbreviations: HCC, hepatocellular carcinoma; OD, optical density; sh, short hairpin.

Figure 3

LINC00339 targeted the miR-1182/SKA1 pathway in HCC. (A) LINC00339 knockdown promoted miR-1182 expression in SMMC-7721 and Hep3B cells. (B) miR-1182 expression was suppressed by LINC00339 overexpression (oe). (C and D) Luciferase reporter assay using wild-type (wt) or mutant (mut) LINC00339 reporter indicated that the activity of wt-LINC00339 was suppressed by miR-1182 mimics. (E and F) RNA pulldown assay showed that biotin-labeled wt-miR-1182 enriched LINC00339 in HCC cell lysates. (G) Analysis of correlation between LINC00339 and miR-1182 in tumor tissues. (H and I) Luciferase reporter assay illustrated that the activity of wt-SKA1 3’-UTR was suppressed by miR-1182 mimics. (J) miR-1182 mimics suppressed the expression of SKA1 in HCC cells. (K) Luciferase reporter assay showed that LINC00339 knockdown suppressed the activity of wt-SKA1 3’-UTR. (L) LINC00339 knockdown suppressed the expression of SKA1 in HCC cells. *p<0.05. All experiments were repeated three times. Abbreviations: HCC, hepatocellular carcinoma; miR, microRNA; sh, short hairpin.

Figure 4

SKA1 restoration rescued the effects of LINC00339 knockdown. (A) TCGA data indicated that SKA1 was upregulated in HCC tissues. (B) qRT-PCR confirmed that SKA1 expression was increased in HCC tissues. (C) TCGA data indicated that SKA1 upregulation in HCC patients predicted a low survival rate. (D) Relative expression of SKA1 in indicated HCC cells. (E–H) CCK8, EdU, and Transwell assays showed that restoration of SKA1 rescued the proliferation, migration, and invasion of HCC cells transfected with LINC00339 shRNA. *p<0.05 and ***p<0.001. All experiments were repeated three times. Abbreviations: HCC, hepatocellular carcinoma; qRT-PCR, quantitative reverse transcription PCR; TCGA, The Cancer Genome Atlas.