Suppression of Heterogeneous Nuclear Ribonucleoprotein C Inhibit Hepatocellular Carcinoma Proliferation, Migration, and Invasion via Ras/MAPK Signaling Pathway

Abstract

Hepatocellular carcinoma (HCC), the most common malignant tumor, has high fatality and recurrence rates. Accumulating evidence shows that heterogeneous nuclear ribonucleoprotein C (HNRNPC), which is mainly involved in RNA splicing, export, and translation, promotes progression and metastasis of multiple tumor types; however, the effects of HNRNPC in HCC are unknown. In the present study, high levels of HNRNPC were detected in tumor tissues compared with para-tumor tissues by immunohistochemical and western blot assays. Furthermore, Cox proportional hazards regression models, the Kaplan-Meier method, and clinicopathologic features analysis showed that HNRNPC was not only an independent prognostic factor for both overall and disease-free survival in HCC but also a predictor of large tumor size and advanced tumor stage. Functional experiments revealed that silencing of HNRNPC not only led to arrest of more HCC cells at G0/G1 phase to inhibit their proliferation, but also suppressed EMT process to block their invasion, and migration in vitro; this was related to the Ras/MAPK signaling pathway. In addition, blocking of HCC cell proliferation regulated by HNRNPC silencing was observed in vivo. Finally, rescue tests showed that after recovery of Ras/MAPK signaling pathway activity by treatment with Ras agonists, the proliferation, migration, and invasion suppression of Huh-7 and Hep 3B cell lines caused by HNRNPC knockdown was partially reversed. Taken together, these results indicate that HNRNPC knockdown inhibits HCC cell proliferation, migration and invasion, in part via the Ras/MAPK signaling pathway. Thus, HNRNPC may have an important role in the progression of HCC and represents a promising biomarker for evaluation of prognosis and a potential therapeutic target in HCC patients.

Keywords: HCC; HNRNPC; MAPK; Ras; alternative splicing.

Figure 1

High HNRNPC expression was detected in HCC tumor tissues, and predicts a poor survival in HCC patients. (A) HNRNPC mRNA expression in HCC tissues and normal liver tissues acquired from UALCAN analysis. (B) UALCAN analysis showing HNRNPC mRNA expression at different cancer stages. (C, D) Western blot showing protein expression of HNRNPC in HCC tumor tissues and matched para-tumor tissues. (E) Classical cases showing HNRNPC protein expression in HCC tumor tissues and paired para-tumor tissues. (F) The χ2 test was used to assess HNRNPC protein expression in HCC tissues and adjacent tissues. (G) Kaplan–Meier analysis showing the correlation of HNRNPC level with overall survival of HCC patients. (H) Kaplan–Meier analysis showing the association between HNRNPC level and disease-free survival. The final results were presented as mean ± standard deviation (SD). *P < 0.05, ^***P < 0.001.

Figure 2

Knockdown of HNRNPC suppressed proliferation, invasion, and migration of HCC cells in vitro. (A) Colony formation assay showing the impact of HNRNPC knockdown on HCC cell proliferation. (B) CCK-8 test showing the effects of HNRNPC downregulation on HCC cell viability. (C) Transwell assay showing the influence of HNRNPC inhibition on HCC cell invasion. (D) Scratch wound test showing the influence of HNRNPC suppression on HCC cell migration. All experiments were independently repeated three times, and the results were showed as mean ± standard deviation (SD). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 3

HNRNPC suppression inhibited the Ras/MAPK signaling pathway, leading to arrest of more cells at S phase and blocking of EMT in vitro. (A, B) Western blot to detect activation of the Ras/MAPK signaling pathway in Lv-NC Huh-7, Lv-HNRNPC Huh-7, Lv-NC Hep 3B, and Lv-HNRNPC Hep 3B cells. (C, D) Cell cycle analysis for the various cell groups mentioned above. (E, F) Western blot to detect c-Myc, CDK4, and cyclin E1 levels in the various cell groups. (G, H) Western blot to analyze Vimentin and E-Cadherin expression in various cell groups. Each test was performed independently in triplicate, and the results were presented as mean ± standard deviation (SD). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 4

HNRNPC knockdown blocked HCC cell proliferation in vivo. (A) Huh-7-cell xenograft tumors in Lv-HNRNPC and Lv-NC groups. (B, C) t-test analysis showing tumor weights and sizes in Lv-HNRNPC and Lv-NC Huh-7 cell groups. (D) H&E staining to confirm xenograft tumors (upper panel); IHC to detect HNRNPC levels in xenograft tumors (middle panel); proliferation of xenograft tumors as determined by Ki-67 staining (nether panel). (E) IHC staining to analyze CD34 (upper panel), Vimentin (middle panel), and E-Cadherin (nether panel) expression in xenograft tumors. The tumor size and weight were demonstrated as mean ± standard deviation (SD). ***P < 0.001.

Figure 5

After treatment with ML-098 (20 nmol/l), MAPK signaling pathway activation was recovered completely in Lv-HNRNPC-group cells, and proliferation ability was partially reversed. (A, B) Western blot to detect recovery of activation of the MAPK signaling pathway in Lv-HNRNPC groups after treatment with ML-098. (C, D) Colony formation assay showing the reversed efficiency of ML-098 in Lv-HNRNPC group cell proliferation. (E) CCK-8 test showing the rescue by ML-098 of cell viability in the Lv-HNRNPC group. The tests mentioned above were operated three times respectively, and the results were reported as mean ± standard deviation (SD). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, for Lv-NC+DMSO group compared with Lv-HNRNPC+ML-098 group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, for Lv-HNRNPC+DMSO group compared with Lv-HNRNPC+ML-098 group.

Figure 6

ML-098 partly reversed the reduction in numbers of S phase cells in the Lv-HNRNPC group. (A, B) Cell cycle analysis to detect the recovery of numbers of S phase cells in the Lv-HNRNPC groups after treatment with ML-098. (C, D) Western blot to detect the recovery of c-Myc, CDK4, and cyclin E1 in Lv-HNRNPC cell groups after treatment with ML-098. All tests were repeated independently in triplicate, and the results were showed as mean ± standard deviation (SD). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, for Lv-NC+DMSO group compared with Lv-HNRNPC+ML-098 group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, for Lv-HNRNPC+DMSO group compared with Lv-HNRNPC+ML-098 group.

Figure 7

ML-098 recovered the invasion and migration abilities of Lv-HNRNPC group cells in part. (A, B) Transwell assay demonstrating the recovered efficiency of ML-098 in Lv-HNRNPC group cell invasion. (C, D) Scratch wound test showing the rescue by ML-098 of cell migration in the Lv-HNRNPC group. (E, F) Western blot to detect the recovery of EMT process in Lv-HNRNPC cell groups after treatment with ML-098. Each test was repeated independently in triplicate, and the results were demonstrated as mean ± standard deviation (SD). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, for Lv-NC+DMSO group compared with Lv-HNRNPC+ML-098 group; ^#P < 0.05, ^##P < 0.01, for Lv-HNRNPC+DMSO group compared with Lv-HNRNPC+ML-098 group.