lncRNA NEAT1 promotes the proliferation and metastasis of hepatocellular carcinoma by regulating the FOXP3/PKM2 axis

Figure 1. Knockdown of NEAT1 can inhibit the viability, proliferation, migration, and invasion of hepatocellular carcinoma cells. (A) qPCR was used to detect the overexpression or interference efficiency of NEAT1. (B, C) The MTT and colony formation assay were used to detect cell viability and proliferation. (D, E) A Transwell assay was performed to evaluate the migration and invasion of HCC cells. ** indicates P <0.05 compared with pcDNA3.1 or vector; ## indicates P <0.05 compared with Si-nc.

Figure 2. NEAT1 binds FOXP3 to promote PKM2 transcription. (A) RNA pulldown, silver staining, and mass spectrometry analysis were carried out to investigate the interaction between NEAT1 and FOXP3. (B) RNA pulldown was performed to verify the binding between NEAT1 and FOXP3. (C, D) RIP experiments confirmed the binding between NEAT1 and FOXP3. (E) Bioinformatics predicted the binding sites of NEAT1 and FOXP3, and RIP again found that NEAT1 was the binding site in the 900–1,200 fragment of FOXP3. (F) Bioinformatics catRAPID predicted the combination score of NEAT1 and FOXP3, proving that the possibility of the combination was high. (G, H) After overexpression or knockdown, qPCR was used to assess the expression of NEAT1 in the cytoplasm and nucleus. (I) A luciferase reporter experiment was performed to investigate whether NEAT1 overexpression or knockdown could change the transcription activity of PKM2. (J) A ChiP assay was performed to detect the interaction between FOXP3 and PKM2 under NEAT1 knockdown or overexpression. (K) qPCR was used to assess the expression of FOXP3 in the plasma and nucleus. (L) A luciferase reporter experiment was performed to investigate the transcription activity…

Figure 3. PKM2 is highly expressed in HCC, and knockdown of PKM2 can inhibit the viability, proliferation, migration, and invasion of HCC cells. (A, B) The expression of PKM2 based on the GSE6764 and GSE14520 datasets from the GEO database was analyzed. (C) The expression of PKM2 in the tissues was detected using qPCR. (D) Western blot was used to detect PKM2 expression in tumor tissues. (E) Survival analysis confirmed that PKM2 was associated with the prognosis of HCC. (F) qPCR was performed to detect PKM2 after overexpression or knockdown. (G) Cell viability was assessed using the MTT assay. (H) Clone formation detection of proliferation was carried out. (I, J) A Transwell assay was carried out to evaluate cell migration and invasion. * indicates P <0.05; ** indicates P <0.01.

Figure 4. PKM2 silencing reversed the function of NEAT1. (A) qPCR was used to detect PKM2 expression. (B) The MTT assay for viability detection was performed. (C) A clone formation assay was used to detect proliferation. (D, E) A Transwell assay was carried out to evaluate cell migration and invasion. ** indicates P <0.01.

Figure 5. In-vivo experiments were conducted to investigate the effects of NEAT1 and PKM2 on the growth of HCC. (A) A picture of the tumor is shown. (B) The curve of tumor growth. (C) The weight of the tumors was assessed. (D) Histochemistry was used to detect the expression of Ki67 and PKM2 in the tumor tissues, proving the conclusion of the cell part. (E, F) Lung metastasis was analyzed. * indicates P <0.05; ** indicates P <0.01.