Paradoxical overexpression of MBNL2 in hepatocellular carcinoma inhibits tumor growth and invasion

Abstract

Pre-mRNA alternative splicing is an essential step in the process of gene expression. It provides cells with the opportunity to create various protein isoforms. Disruptions of alternative splicing are associated with various diseases, including cancer. The muscleblind-like (MBNL) protein is a splicing regulatory protein. Overexpression of MBNL proteins in embryonic stem cells promotes differentiated cell-like alternative splicing patterns. We examined the expression level of MBNL2 in 143 resected HCCs using immunohistochemistry. MBNL2 was overexpressed in 51 (35.7%) HCCs. The overexpression of MBNL2 correlated with smaller tumor size (≤ 3 cm, P = 0.0108) and low tumor stage (Stage I, P = 0.0026), indicating that MBNL2 expression was lost in the late stage of HCC development. Furthermore, patients with MBNL2-positive HCCs had a borderline better 5-year overall survival (P = 0.0579). In non-cancerous liver parenchyma, MBNL2 was stained on the Canals of Hering and hepatocytes newly derived from hepatic progenitor cells. The overexpression of MBNL2 in Hep-J5 cells suppressed proliferation, tumorsphere formation, migration, and in vitro invasion, and also reduced in vivo tumor growth in NOD/SCID mice. In contrast, MBNL2 depletion with RNA interference in Huh7 cells increased in vitro migration and invasion, but did not enhance tumor growth. These results indicate that MBNL2 is a tumor suppressor in hepatocarcinogenesis.

Keywords: alternative splicing; hepatic progenitor cells; hepatocellular carcinoma; liver carcinogenesis; muscleblind proteins.

Figure 1. Immunostaining of MBNL2 in non-cancerous liver parenchyma and HCC

A. In the non-cancerous part, MBNL2 was expressed in the bile ducts (arrow) and canals of Hering (arrowhead). B. In occasional specimens, MBNL2 was expressed in the compressed liver parenchyma (* area) adjacent to the tumor nodule. C, D. MBNL2 stained both cytoplasm and nuclei of HCC tumor cells. In most specimens, it demonstrated heterogeneous staining (C), but diffuse positivity was observed in some specimens (D). A, B, C, D x 200 (original magnification).

Figure 2. Kaplan–Meier analysis of 5-year overall survival (OS) in 143 patients with HCC

The HCCs with MBNL2 protein expression correlated with borderline higher OS than the HCCs without MBNL2 protein expression (P = 0.0579).

Figure 3. Expression of MBNL2 in hepatocytes newly derived from hepatic progenitor cells

A. Colocalization of MBNL2 and EpCAM in the non-cancerous liver parenchyma in serial sections indicates that MBNL2 protein was induced in hepatocytes newly derived from hepatic progenitor cells. B. Ki-67 immunostaining confirmed regeneration of hepatocytes after two-thirds partial hepatectomy (PH) in C57BL/6 mice. The expression of Ki-67 in day 3 post-PH was significantly higher than the expression of Ki-67 in day 1 post-PH. C. Western blot analysis showed a gradual induction of MBNL2 protein expression up to Day 7 post-PH in C57BL/6 mice. D. Immunostaining also demonstrated that the MBNL2-positive cells increased gradually post-PH, and nearly all hepatocytes were positive for MBNL2 on Day 7 post-PH in C57BL/6 mice. ***P value < 0.001, Student's test.

Figure 4. Overexpression of MBNL2 suppressed proliferation and invasion in Hep-J5 HCC cell line

A. Western blot analysis confirmed the overexpression of MBNL2 protein in Hep-J5 cells through lentiviral transduction. B. Cell-counting assay indicated that overexpression of MBNL2 did not affect the anchorage-dependent cell growth. C. Soft agar assay demonstrated that overexpression of MBNL2 significantly suppressed the anchorage-independent cell growth. D. Tumorigenicity assay showed that overexpression of MBNL2 significantly suppressed the in vivo growth of the subcutaneous xenograft in NOD/SCID mice. E, F. Overexpression of MBNL2 also suppressed the migration (E) and invasion (F) ability of Hep-J5 cells significantly. (*P value < 0.05; **P value < 0.01; ***P value < 0.001, Student's test).

Figure 5. Knockdown of MBNL2 enhanced the invasion ability of HCC

A. Western blot analysis showed that short hairpin RNAs (shRNA) #1~#4 markedly reduced the protein expression of MBNL2 in Huh7 cells. B. Cell-counting assay showed that the proliferation rates of parental cells, vector control, and cells with MBNL2 knockdown by shRNA #1 and #2 were similar. C. Soft agar assay indicated that MBNL2 knockdown did not alter the anchorage-independent growth ability in Huh7 cells. D, E. Boyden chamber assay showed that knockdown of MBNL2 by shRNA #1 and #2 significantly enhanced the migration (D) and invasion (E) ability in Huh7 cells. (**P value < 0.01; ***P value < 0.001, Student's test.)

Figure 6. Overexpression of MBNL2 suppressed the formation of tumorspheres and expression of stem cell markers

A. Overexpression of MBNL2 through lentiviral transduction.in Hep-J5 cells reduced the tumorsphere formation in the suspension culture. B, C, D. Quantitative real-time PCR assay demonstrated that overexpression of MBNL2 in Hep-J5 cells through lentiviral transduction significantly suppressed the expression of stem cell markers SOX2 (B), NANOG (C), and OCT4 (D). (*P value < 0.05; **P value < 0.01, Student's test.)