Bacterial metabolite butyrate in modulating sorafenib-targeted microRNAs to curtail its resistance in hepatocellular carcinoma

Abstract

Background and aim: The host dietary fibre is fermented into short-chain fatty acids (SCFA) by intestinal microbiota as bacterial metabolites like propionate, acetate and butyrate. Among these metabolites, the role of butyrate is well documented to provide energy to intestinal epithelial cells. Also, butyrate has anti-inflammatory and anti-tumour properties and decrease in its level by unbalanced diet can develops cancer. Lately, some research has suggested that sodium butyrate as an inhibitor of histone deacetylase (HDAC) may have anticancer potential for hepatocellular carcinoma (HCC), the most common type of liver cancer. Since, HCC is asymptomatic it is usually diagnosed at its advanced stage. Sorafenib with antiproliferative and antiangiogenic effects is the first line of treatment in advanced HCC. However, prolonged drug treatment to HCC patients develops adaptive resistance towards the sorafenib. Sorafenib resistance can also be enhanced by differentially expressed microRNAs. However, the significance of butyrate in HCC sorafenib resistance and its association with sorafenib-targeted microRNAs is yet to be unfurled. Here, an attempt has been made to explore the role of bacterial metabolite butyrate on sorafenib resistant HCC as well as on sorafenib-targeted microRNAs (miR-7641 and miR-199) to curtail sorafenib resistance in HCC.

Methods: Initially, in-silico analysis was performed using Human Metabolome Database (HMDB) so to identify specific butyrate producing faecal bacteria. Then, their specific 16s rRNA expression was compared between HCC patients and healthy individuals using qRT-PCR. Additionally, the cell viability (MTT) and apoptosis assays were performed in both parental and sorafenib resistant HepG2 cells to evaluate the role of sodium butyrate in sorafenib resistant HCC. Moreover, the association of sodium butyrate with sorafenib-targeted miR-7641 and miR-199 was also assessed using real time PCR, cell viability, cell apoptosis and transfection assays.

Results: In silico analysis demonstrated Roseburia cecical, Roseburia intestinalis, Eubacterium rectal, Faecalibacterium prausnitzii as specific butyrate producing faecal bacteria and their 16s rRNA expression was downregulated in HCC patients. In vitro study revealed the presence of sodium butyrate also decreased the cell viability as well as enhanced cell apoptosis of both parental and resistant HepG2 cells. Interestingly, sodium butyrate also decreased the expression of both sorafenib-targeted miR-7641 and miR-199. Further, combination of both sodium butyrate and antimiR-7641 or antimiR-199 also increased apoptosis and decreased viability of resistant cells.

Conclusion: This is first study to unravel the association of butyrate producing bacteria with HCC patients and the significance of bacterial metabolite butyrate as anti-tumour in sorafenib resistant hepatocellular carcinoma. The study also demonstrated the plausible new aspects of bacterial metabolite butyrate association with sorafenib-targeted miRNAs (miR-7641 and miR-199). Hence, the study highlighted the therapeutic potential of bacterial metabolite butyrate that might improve the clinical management of hepatocellular carcinoma.

Keywords: Bacterial metabolite butyrate; Carcinoma; Hepatocellular; MicroRNAs; Resistance; Sorafenib.

Fig. 1

HMDB database shows A Butyrate as bacterial metabolite. B Eubacterium, Roseburia and Faecalibacterium as butyrate producing bacteria

Fig. 2

HMDB shows butyrate as bacterial metabolite in A Roseburia intestinalis. B Eubacterium rectale. C Roseburia cecical. D Faecalibacterium prausnitzii

Fig. 2

HMDB shows butyrate as bacterial metabolite in A Roseburia intestinalis. B Eubacterium rectale. C Roseburia cecical. D Faecalibacterium prausnitzii

Fig. 3

Quantitation of 16S rRNA of A Roseburia intestinalis. B Eubacterium rectale. C Roseburia cecical. D Faecalibacterium prausnitzii

Fig. 4

Sorafenib resistant HepG2 cells characterization A Morphological analysis. B Dose–Response Curve (IC50 = 2.3) HepG2 (P); parental and HepG2 (R); sorafenib resistant

Fig. 5

Morphological analysis of HepG2 cells in presence of increasing concentration of sodium butyrate A Control (Without treatment) B 0.5 mM sodium butyrate C 1 mM sodium butyrate D 3 mM sodium butyrate E 5 mM sodium butyrate F 10 mM sodium butyrate

Fig. 5

Morphological analysis of HepG2 cells in presence of increasing concentration of sodium butyrate A Control (Without treatment) B 0.5 mM sodium butyrate C 1 mM sodium butyrate D 3 mM sodium butyrate E 5 mM sodium butyrate F 10 mM sodium butyrate

Fig. 6

Cell viability of HepG2 cells in presence of increasing concentration of sodium butyrate

Fig. 7

Cell apoptosis comparing A Parental and sorafenib resistant cells B presence of increasing concentration of sodium butyrate

Fig. 8

HepG2 cells treatment with A miR-7641 B AntimiR-7641 C miR-199 D AntimiR-199

Fig. 8

HepG2 cells treatment with A miR-7641 B AntimiR-7641 C miR-199 D AntimiR-199

Fig. 9

Cell apoptosis in presence of miRs and AntimiRs A Flow cytometry representation B Bar graph representation

Fig. 10

Effect of sodium butyrate on miR-7641 expression

Fig. 11

Effect of sodium butyrate on miR-199 expression

Fig. 12

A 96-well plate showing MTT assay to estimate cell viability (i) Control; (ii) AntimiR-7641 (30 nM); (iii) AntimiR-199 (30 nM); (iv) AntimiR-7641 + sodium butyrate (5 mM); (v) AntimiR-199 + sodium butyrate (5 mM); (vi) AntimiR-7641 + sorafenib (2 µM) + sodium butyrate (5 mM); (vii) AntimiR-199 + sorafenib (2 µM) + sodium butyrate (5 mM). (B) Bar graph representation of cell viability in presence of different treatments (i) Control; (ii) AntimiR-7641 (30 nM); (iii) AntimiR-199 (30 nM); (iv) AntimiR-7641 + sodium butyrate (5 mM); (v) AntimiR-199 + sodium butyrate (5 mM); (vi) AntimiR-7641 + sorafenib (2 µM) + sodium butyrate (5 mM); (vii) AntimiR-199 + sorafenib (2 µM) + sodium butyrate (5 mM)

Fig. 13

Bar graph representation of cell apoptosis in presence of different treatments (i) Control; (ii) AntimiR-7641 (30 nM); (iii) AntimiR-199 (30 nM); (iv) AntimiR-7641 + sodium butyrate (5 mM); (v) AntimiR-199 + sodium butyrate (5 mM); (vi) AntimiR-7641 + sorafenib (2 µM) + sodium butyrate (5 mM); (vii) AntimiR-199 + sorafenib (2 µM) + sodium butyrate (5 mM)