Let-7a enhances the sensitivity of hepatocellular carcinoma cells to cetuximab by regulating STAT3 expression

Figures & data

Figure 1 Let-7a can enhance the sensitivity of HCC cells with epithelial phenotypes to cetuximab.

Notes: (A–E) All HCC cell lines were separated into three groups: the let-7a mimic, inhibitor, and control groups. Cell viability was determined using the CCK-8 assay in the presence of different concentrations of cetuximab (0, 500, 1,000, 1,500, and 2,000 µg/mL). Cell proliferation rate (using the EdU assay) was determined in the presence of an IC₅₀ concentration (Huh7: 2,564 µg/mL, Hep3B: 2,299 µg/mL, HepG2: 7,167 µg/mL, SNU449: 2,450 µg/mL, and SNU387: 3,182 µg/mL) of cetuximab (^*P<0.05, ^**P<0.01 vs control. Huh7: P_{Let-7a mimic+Cet}=0.0284, P_{Let-7a inhibitor+Cet}=0.0378; Hep3B: P_{Let-7a mimic+Cet}=0.0051, P_{Let-7a inhibitor+Cet}=0.0218; HepG2: P_{Let-7a mimic+Cet}=0.0415, P_{Let-7a inhibitor+Cet}=0.0138).
Abbreviations: CCK-8, Cell Counting Kit-8; Cet, cetuximab; IC₅₀, half maximal inhibitory concentration; HCC, hepatocellular carcinoma.

Table 1 IC₅₀ values for cetuximab in HCC cell lines with or without let-7a mimic or let-7a inhibitor treatment

HCC cell line	IC50 (μg/mL)
	Cetuximab	Cetuximab + let-7a mimic	Cetuximab + let-7a inhibitor
Huh7	2,564±109.8	2,104±96.51*	3,853±495.5*
Hep3B	2,299±76.91	1,279±44.54*	3,000±287.3*
HepG2	7,167±2293	2,732±170.5*	7,913±3625*
SNU387	3,182±190.9	2,814±216.9	2,473±137.0
SNU449	2,450±70.66	2,406±99.81	2,511±157.8

Notes:

* P<0.05 vs cetuximab alone. Data presented as mean ± SD.

Abbreviations: HCC, hepatocellular carcinoma; IC₅₀, half maximal inhibitory concentration; SD, standard deviation.

Figure 2 Let-7a negatively regulates the expression of STAT3.

Notes: (A) Predicted matching miRNAs for the STAT3 3′-UTR segment by TargetScan analysis. (B) mRNA expression of STAT3 and let-7a in the various HCC cell lines. (C) mRNA expression of STAT3 in the various HCC cell lines in the presence of the miRNA let-7a mimic, inhibitor, and control. (D) mRNA expression of let-7a in the various HCC cell lines in the presence of the miRNA let-7a mimic, inhibitor, and control. (E) Protein expression of STAT3 in the various HCC cell lines in the presence of the miRNA let-7a mimic, inhibitor, and control as detected by western blot (^*P<0.05, ^**P<0.01, ^***P<0.001, Huh7: P_{Let-7a mimic+Cet}=0.0338, P_{Let-7a inhibitor+Cet}=0.0047; Hep3B: P_{Let-7a mimic+Cet}=0.0080, P_{Let-7a inhibitor+Cet}=0.0289; HepG2: P_{Let-7a mimic+Cet}=0.0004, P_{Let-7a inhibitor+Cet}=0.0206; SNU387: P_{Let-7a mimic+Cet}=0.0003, P_{Let-7a inhibitor+Cet}=0.0062; SNU449: P_{Let-7a mimic+Cet}=0.0022, P_{Let-7a inhibitor+Cet}=0.0054).
Abbreviations: HCC, hepatocellular carcinoma; miRNA, microRNA; mRNA, messenger RNA; STAT3, signal transducer and activator of transcription 3.

Figure 3 Suppression of STAT3 could enhance the sensitivity of epithelial phenotype HCC cells to cetuximab.

Notes: (A) Transfection efficiency of STAT3 siRNA detected by Western blot in the five HCC cell lines; GAPDH was used as control (^*P<0.05, ^**P<0.01, ^***P<0.001 Huh7: P=0.0353; Hep3B: P=0.0005; HepG2: P=0.0067; SNU387: P=0.0001; SNU449: P=0.0002). (B–F) Cell viability was determined using the CCK-8 assay in the presence of different concentrations of cetuximab (0, 500, 1,000, 1,500, and 2,000 µg/mL). All HCC cell lines were separated into two groups: a STAT3 siRNA group and a negative siRNA control group.
Abbreviations: CCK-8, Cell Counting Kit-8; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3.

Table 2 IC₅₀ values for cetuximab in HCC cell lines with negative siRNA plus cetuximab treatment or cetuximab plus STAT3 siRNA treatment

HCC cell line	IC50 (μg/mL)
	Negative siRNA + cetuximab	STAT3 siRNA + cetuximab
Huh7	2,564±109.8	2,037±134.7*
Hep3B	2,167±68.96	1,562±74.38*
HepG2	7,167±2,293	1,680±79.01*
SNU387	2,554±154.2	2,487±272.2
SNU449	2,220±71.31	2,239±115.1

Notes:

* P<0.05 vs negative siRNA + cetuximab. Data presented as mean ± SD.

Abbreviations: HCC, hepatocellular carcinoma; IC₅₀, half maximal inhibitory concentration; SD, standard deviation; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3.

Figure 4 The role of let-7a on increasing the sensitivity of HCC cells to cetuximab is impaired after STAT3 suppression.

Notes: (A–E) After STAT3 knockdown, the HCC cell lines were separated into three groups, and separately transfected with the let-7a mimic, inhibitor, or control. Cell viability was determined using the CCK-8 assay in the presence of different concentrations of cetuximab (0, 500, 1,000, 1,500, and 2,000 µg/mL).
Abbreviations: CCK-8, cell counting kit-8; HCC, hepatocellular carcinoma; STAT3, signal transducer and activator of transcription 3.