Effect of TALEN-mediated IL-6 knockout on cell proliferation, apoptosis, invasion and anti-cancer therapy in hepatocellular carcinoma (HCC-LM3) cells

Abstract

Purpose: To determine the exact effect of Interleukin-6 (IL-6) on tumor cell proliferation, apoptosis, invasion, and anti-cancer therapy in hepatocellular carcinoma (HCC).

Experimental design: IL-6 was disrupted by transcription activator-like effector nucleases (TALEN) in HCCLM3 cells, and was used to evaluate the role of IL-6 on tumor cell proliferation, apoptosis, invasion and key signaling pathways involved in sorafenib and/or IFNα therapy.

Results: IL-6 has no direct effect on cell proliferation and invasion but promotes cell apoptosis and up-regulate IL-33 and VEGF-A expression. IL-6 could attenuate the anti-proliferation effect by sorafenib and combination therapy but facilitate the pro-apoptosis of the combination therapy and augment the pro-invasive effect induced by single treatment. IL-6 could down-regulate p-STAT3, however up-regulate the p-MEK/p-ERK and NF-kB/iNOS expression, and it also facilitated the promotion on p-JAK2 and p-MEK/p-ERK by either sorafenib or IFN-α. in vivo study, IL-6 significantly promotes tumor growth. The combination treatment showed the highest inhibition on tumor growth which is derived from HCCLM3-IL6(-) cells.

Conclusions: IL-6 has no direct effect on cell proliferation and invasion but promotes tumor cell apoptosis in vitro study. Sorafenib and combination therapies are suitable for HCC cells with low or no IL-6 expression confirmed in vivo study.

Keywords: IL-6; TALEN; hepatocellular carcinoma.

Figure 1. Stable cell line construction using TALENs

(A) The TALEN design is in accordance to the sequence of IL-6. The arms of TALEN were designed as a 2×3 (2 left arms and 3 right arms) combination targets on the IL-6 (NCBI gene ID: 3569). The plasmids for the left and right arms of the TALENs were constructed using the FAST TALEN Kit (SIDANSAI, China). (B) After sequencing, five plasmids were transfected into HEK 293T cell lines using FuGene HD transfection reagent (Roche) in a 2×3 cross combination. A pair of TALEN (L2R3) plasmids was selected as the most effective knockout group after 3 days of puromycin screening and subsequent genomic PCR sequencing. (C) Mono-clone 25 exhibited bi-allelic IL-6 mutations. One allelic IL-6 was deleted at 5 bp, and the other was deleted at 7 bp on the same region.

Figure 2. IL-6 knock-out had no effect on cell proliferation but enhanced the anti-proliferation effect by sorafenib and combination therapy

No significant difference was observed in the proliferation between HCCLM3-wt and HCCLM3-IL6(-) cells for 24 and 48 hr. However, the proliferation of HCCLM3-wt cells could not be significantly inhibited by IFN-α and inversely inhibited by sorafenib. The inhibitory effect was not distinctly enhanced by the co-treatment of IFN-α and sorafenib. IL-6 attentuated the anti-proliferative effect of sorafenib as well as the co-treatment of sorafenib and IFN-α for 24 and 48 hr. Cell proliferation was evaluated by CCK-8 assay.

Figure 3. IL-6 knock-out attenuated side pro-invasive effect induced by the single treatment of either sorafenib or IFN-α

IL-6 had no effect on tumor invasion; the cell invasion capacity was significantly increased in 24 and 48 hr by single treatment of either sorafenib or IFN-α treatment, which was not observed in the co-treatment group; IL-6 knock-out could attenuate the pro-invasive side effect of single treatment; however the effect was not observed when co-treatment was conducted.

Figure 4. IL-6 promotes tumor cells apoptosis and enhance the pro-apoptosis effect by combining sorafenib and IFN-α

(A-C) The cell apoptosis rate of HCCLM3-wt cells was significantly higher than that of HCCLM3-IL6(-) cells for 24 and 48 hr. No significant effect on cell apoptosis was found by either sorafenib or IFN-α in both cell lines. However, the prominently increased cell apoptosis by co-treatment was observed and was significantly attenuated in HCCLM3-IL6(-) cells. (D-E) Higher NO levels was observed in HCCLM3-wt cells as compared with HCCLM3-IL6(-) cells, and highest was observed in combination treatment.

Figure 5. IL-6 knock-out down-regulate the tumor derived IL-33 and VEGF-A expression

The supernatant of tumor cell culture was assessed by using ELISA assay. We found that the IL-4, TNF-α, MCP-1, PDGF-BB levels was similar in both 2 cell lines. However, IL-33 and VEGF-A were significantly decreased in the HCCLM3-IL6(-) cells for 24 hr and 48 h, respectively.

Figure 6. IL-6 on signaling transduction pathways including p-MEK/p-ERK, p-JAK2/p-STAT3, and NF-kB-iNOS

Three major signaling transduction pathways, including p-MEK/p-ERK, p-JAK2/p-STAT3, and NF-kB-iNOS were tested to explore the downstream modulation of IL-6. IL-6 could down-regulate the protein expression of p-STAT3 in a time-dependent manner, However, IL-6 has no effect on p-JAK2 expression. IL-6 has a promotion effect on the expression of p-MEK/p-ERK and NF-kB/iNOS. Furthermore, other proliferation- and apoptosis-related protein, such as cyclin-D1 and Bcl-2, were not affected by the IL-6. We found that the p-STAT3 expression was similarly reduced by the sorafenib in both cell lines in 24 hr and significantly reduced in 48 hr in HCCLM3-wt cells. p-JAK2, p-MEK and p-ERK were inversely up-regulated by sorafenib in a time-dependent manner. IFN-α had the opposite promotion effect on p-JAK2/p-STAT3 and p-MEK/p-ERK. The effect of co-treatment on these three major signaling transduction pathways is similar as the sorafenib group in terms of p-JAK2/p-STAT3 and p-MEK/p-ERK. In terms of NF-kB/iNOS, either IFN-α or sorafenib has no significant effect on the cell signal pathways. However, both statistically increased in the combination group in HCCLM3-wt cells in 48 hr.

Figure 7. IL-6 on tumor growth and treatment in vivo

IL-6 significantly promote tumor growth in vivo study for six wk as compared with HCC with HCCLM3-IL6(-) cells. Sorafenib has predominant inhibition on tumor growth when IL-6 was knocked out, whereas IFN-α treatment significantly inhibited tumor growth in the HCC derived from HCCLM3-wt cells. The combination of sorafenib and IFN-α treatment showed the highest predominant inhibition on tumor derived from HCCLM3-IL6(-) cells. (A) Control group (HCCLM3-wt); (B) control group (HCCLM3-IL-6(-)); (C) IFN-α treatment group (HCCLM3-wt); (D) IFN-α treatment group (HCCLM3-IL-6(-)); (E) sorafenib treatment group (HCCLM3-wt); (F) sorafenib treatment group (HCCLM3-IL-6(-)); (G) combination treatment group (HCCLM3-wt); (H) combination treatment group (HCCLM3-IL-6(-)).