Long non-coding RNA LOC107985656 represses the proliferation of hepatocellular carcinoma cells through activation of the tumor-suppressive Hippo pathway

Abstract

Long non-coding RNAs (lncRNAs) play important regulatory roles in hepatocellular carcinoma (HCC). However, the function of LOC107985656 in HCC progression remains unclear. The lncRNA, mRNA and miRNA levels in HCC tissues or cells were measured using real-time quantitative polymerase chain reaction (RT-qPCR). The proliferation of cancer cells was evaluated using 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) viability and colony formation assays. Bioinformatics prediction, dual luciferase assay and RNA pull-down assay were performed to analyze the relationships between LOC107985656 and miR-106b-5p, or miR-106b-5p and large tumor suppressor 1 (LATS1). The protein expression levels were detected using Western blot. Results showed that LncRNA LOC107985656 was downregulated in HCC tissues and cells. Upregulation of LOC107985656 inhibited the proliferation of HCC cells, whereas its knockdown promoted this phenomenon. LOC107985656 could activate the tumor-suppressive Hippo pathway by repressing yes association protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) (two homologs of Yki) protein expression in HCC. Further investigation suggested that LOC107985656 regulated the expression of LATS1 by acting as a sponge for absorbing miR-106b-5p in HCC cells. In conclusion, this study unraveled the role of LOC107985656 following a ceRNA (competing endogenous RNAs) mechanism for the miR-106b-5p/LATS1 axis in HCC. The results indicate potential diagnostic and therapeutic applications of LOC107985656 in HCC.Abbreviations:HCC: hepatocellular carcinoma; LncRNA: long non-coding RNA; LATS1: large tumor suppressor 1; MTT: 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; YAP: yes association protein; WWTR1: WW domain-containing transcription regulator protein 1; cDNA: single-stranded complementary DNA; RT-qPCR: real-time quantitative polymerase chain reaction; Radio-Immunoprecipitation Assay (RIPA); BCA: bicinchoninic acid; ASO: antisense oligonucleotide; MST1/2: Ste20-like kinases 1/2; TEAD: TEA domain transcription factor; ceRNA: competing endogenous RNAs.

Keywords: Lncrna; hcc; hippo pathway; lats1; loc107985656; miR-106b-5p.

Figure 1.

LOC107985656 was downregulated in HCC tissues and cells. (a) Relative expression of LOC107985656 in HCC tissues (n = 30) and corresponding non-tumor tissues (n = 30) according to RT-qPCR results. (b) Kaplan–Meier survival analysis demonstrating that the low expression of LOC107985656 was associated with poor prognosis in patients with HCC. (c) RT‐qPCR results showing that LOC107985656 was downregulated in HCC cell lines (Huh7, SMMC‐7721, HepG2.2.15, and HepG2) compared with immortalized normal human hepatocyte cell line LO2. *p < 0.05, and **p < 0.01

Figure 2.

LOC107985656 inhibited the proliferation of HCC cells. (a) RT‐qPCR of the expression of LOC107985656 in HCC cells after transfection with indicated plasmids. (b) MTT assay of the viabilities of HCC cells induced by LOC107985656 overexpression or knockdown. (c and d) Effects of LOC107985656 overexpression and knockdown on the proliferation of HCC cells as analyzed by colony formation assay. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0 .0001

Figure 3.

LOC107985656 activated the tumor-suppressive Hippo pathway. (a) RT‐qPCR of the mRNA level of YAP and TAZ in HCC cells after transfection with LOC107985656 overexpression and knockdown plasmids. (b) Western blot of YAP, p-YAP, TAZ and p-TAZ expression in HCC cells. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance

Figure 4.

LOC107985656 upregulated the expression of LATS1. (a) RT‐qPCR of the mRNA level of LATS1 in HCC cells after transfection with LOC107985656 overexpression and knockdown plasmids. (b) Western blot assay results of the protein level of LATS1 in HCC cells. (c) RT-qPCR of the relative mRNA level of LATS1 in HCC tissues (n = 30) and corresponding non-tumor tissues (n = 30). (d) Positive correlation between LOC107985656 mRNA levels and LATS1 mRNA levels in HCC tissues. **p < 0.01, ***p < 0.001, and ****p < 0 .0001

Figure 5.

LOC107985656 upregulated the expression of LATS1 by sponging miR-106b-5p. (a) Venn diagram of potential miRNAs that targeted LOC107985656 and LATS1 (overlapping fraction). miRNAs that targeted LOC107985656 and LATS1 were predicted by an online database miRDB (http://mirdb.org/) and TargetScanHuman 7.2 (http://www.targetscan.org/vert_72/), respectively. (b) Predicted binding sites for miR-106b-5p in LOC107985656 and the 3ʹUTR of LATS1 as well as the mutations in the binding sites. (c) Luciferase activity in HCC cells co-transfected with pGL/Luc- LOC107985656 wild type or pGL/Luc-LOC107985656 mut with miR-106b-5p mimics or ASO-miR-106b-5p. (d) Luciferase activity in HCC cells co-transfected with pGL/Luc-LATS1 3ʹUTR wild type or pGL/Luc-LATS1 3ʹUTR mut with miR-106b-5p mimics or ASO-miR-106b-5p. (g and h) RT‐qPCR and Western blot assay of the mRNA and protein levels of LATS1 in HCC cells after transfection with miR-106b-5p mimics or its inhibitors ASO-miR-106b-5p. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0 .0001; ns, no significance

Figure 6.

LOC107985656 activated the Hippo pathway through regulating the miR-106b-5p/LATS1 axis. (a) Western blot of the protein levels of LATS1 and YAP were partly reversed by co-transfection with miR-106b-5p. (b) Proposed molecular mechanism of LOC107985656, which acted as a ceRNA mechanism for the miR-106b-5p/LATS1 axis and activated Hippo pathway to inhibit PTC cell proliferation. **p < 0.01; ***p < 0.001; ****p < 0.0001