Kaempferol inhibits proliferation, migration, and invasion of liver cancer HepG2 cells by down-regulation of microRNA-21.

Liver cancer is one of the most common and lethal cancers in human digestive system, which kills more than half a million people every year worldwide. This study aimed to investigate the effects of kaempferol, a flavonoid compound isolated from vegetables and fruits, on hepatic cancer HepG2 cell proliferation, migration, invasion, and apoptosis, as well as microRNA-21 (miR-21) expression. Cell viability was detected using cell counting kit-8 (CCK-8) assay. Cell proliferation was measured using 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Cell apoptosis was assessed using Guava Nexin assay. Cell migration and invasion were determined using two-chamber migration (invasion) assay. Cell transfection was used to change the expression of miR-21. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to analyze the expressions of miR-21 and phosphatase and tensin homologue (PTEN). Expression of key proteins involved in proliferation, apoptosis, migration, invasion, and phosphatidylinositol 3-kinase/protein kinase 3/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway were evaluated using western blotting. Results showed that kaempferol significantly inhibited HepG2 cell proliferation, migration, and invasion, and induced cell apoptosis. Kaempferol remarkably reduce the expression of miR-21 in HepG2 cells. Overexpression of miR-21 obviously reversed the effects of kaempferol on HepG2 cell proliferation, migration, invasion, and apoptosis. Moreover, miR-21 negatively regulated the expression of PTEN in HepG2 cells. Kaempferol enhanced the expression of PTEN and inactivated PI3K/AKT/mTOR signaling pathway in HepG2 cells. In conclusion, kaempferol inhibited proliferation, migration, and invasion of HepG2 cells by down-regulating miR-21 and up-regulating PTEN, as well as inactivating PI3K/AKT/mTOR signaling pathway.

Introduction

Liver cancer is one of the most common and lethal cancers in human digestive system, which kills more than half a million people every year worldwide.[1,2] According to the different origins, liver cancer can be divided into two major categories: primary liver cancer and secondary liver cancer.[3] Primary liver cancer originates from uncontrolled liver cell proliferation, which is often called as hepatocellular cancer.[4] Secondary liver cancer originates from uncontrolled cell proliferation in other organs, such as lung, stomach, colon, and breast, and metastasizes to liver finally, which is often called as metastasis liver cancer.[5,6] The initial symptoms of liver cancer are not obvious, which limits the early diagnosis and treatment for liver cancer.[7] The clinical symptoms of advanced liver cancer are liver pain, fatigue, weight loss, jaundice, and ascites.[8] It is worthy believing that searching for more effective novel medicines for inhibiting liver cancer cell proliferation and metastasis will be helpful for liver cancer treatment.

As a flavonoid compound isolated from vegetables and fruits, kaempferol has aroused more and more attention worldwide due to its wide range of pharmacological activities, such as anti-inflammatory, anti-oxidant, anti-cancer, anti-diabetic, and cardio-protective activities.[9-13] For liver cancer, Guo et al.[14] indicated that kaempferol induced liver cancer HepG2 and Huh7 cell death through endoplasmic reticulum stress-C/EBP homologous protein (CHOP)-autophagy signaling pathway. Mylonis et al.[15] demonstrated that kaempferol suppressed hypoxia-inducible factor 1 (HIF-1) expression and liver cancer cell viability under hypoxia conditions. More experimental researches are still needed to further explore the effects of kaempferol on liver cancer.

MicroRNAs are a class of small single-stranded RNAs with around 22 nucleotides (nt) in eukaryotic cells.[16] MicroRNA-21 (miR-21) has been found to participate in the proliferation and metastasis of many cancer cells.[17] Pineau et al.[18] proved that miR-21 overexpression contributed to liver tumorigenesis. Zhu et al.[19] revealed that miR-21 played critical roles in the migration and invasion of hepatocellular cancer. Meng et al.[20] indicated that miR-21 modulated the expression of phosphatase and tensin homologue (PTEN) in human hepatocellular cancer. Kim et al.[21] demonstrated that kaempferol suppressed vascular smooth muscle cell migration by regulating bone morphogenetic protein (BMP)-mediated miR-21 expression. However, there is no information available about the effects of kaempferol on miR-21 expression in liver cancer cells.

Therefore, in this research, the effects of kaempferol on liver cancer HepG2 cell proliferation, migration, invasion, and apoptosis, as well as miR-21 expression, were investigated. The possible molecular mechanism and signaling pathway were also elucidated. These findings will be helpful for further understanding the anti-cancer effects of kaempferol on liver cancer.

Materials and methods

Cell culture and treatment

Human liver cancer HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT, USA) and 1% Penicillin–Streptomycin solution (Gibco, Life Technologies). Cells were maintained in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO2.

Preparation of kaempferol solution

Kaempferol was purchased from Sigma–Aldrich (St Louis, MO, USA; catalog number: K0133, isolated from the rhizome of Ginkgo biloba L. with purity >90%) and dissolved in dimethyl sulfoxide (DMSO; Sigma–Aldrich) to a storage concentration of 100 mM according to the manufacturer’s instruction. Then, kaempferol solution was sterilized through 0.22 μm filter and stored at -4°C until use. Serum-free DMEM was used to dilute kaempferol solution to experimental concentration. Chemical structure of kaempferol is shown in Figure 1(a).

Figure 1.

Kaempferol inhibits proliferation and induced apoptosis of HepG2 cells. (a) Chemical structure of kaempferol. (b) Viability of HepG2 cells after 0, 25, 50, 75, or 100 μM kaempferol treatment were measured using cell counting kit-8 (CCK-8) assay. (c) Proliferation of HepG2 cells after 50 μM kaempferol treatment was detected using 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. (d) Expression of Cyclin D1 in HepG2 cells after 50 μM kaempferol treatment was assessed using western blotting. (e) Apoptosis of HepG2 cells after 50 μM kaempferol treatment was determined using Guava Nexin assay. (f) Western blotting was performed to analyze the expressions of pro-caspase 3, cleaved-caspase 3, pro-caspase 9, cleaved-caspase 9, Bcl-2, and Bax in HepG2 cells after 50 μM kaempferol treatment.

*P < 0.05; **P < 0.01; ***P < 0.001.

Cell viability assay

Cell counting kit-8 (CCK-8) assay was performed to detect the viability of HepG2 cells after kaempferol treatment. Briefly, HepG2 cells were seeded, in triplicate, in 96-well plate (Thermo Fisher Scientific) with a density of 1 × 104 cells/well and treated by 25, 50, 75, or 100 μM kaempferol for 24 h. After treatment, 10 μL CCK-8 solution was added into each well of the plate and the cell plate was maintained in humidified incubator at 37°C for 1 h. Then, the absorbance at 450 nm of each well was recorded using microplate reader (BioTek Instruments, Winooski, VT, USA). Cell viability (%) was calculated as follows: average absorbance of kaempferol treatment group/average absorbance of control group × 100%.

Cell proliferation assay

Proliferation of HepG2 cells after kaempferol treatment and/or miR-21 mimic transfection were measured using 5-bromo-2′-deoxyuridine (BrdU) incorporation assay kit (Sigma–Aldrich) in line with the manufacturer’s protocol. Briefly, HepG2 cells were seeded, in triplicate, in 6-well plate (Thermo Fisher Scientific) with a density of 1 × 105 cells/well. BrdU solution was added into each well of the plate before 50 μM kaempferol treatment by 4 h. After kaempferol incubation for 24 h, BrdU positive(+) cells in each well was counted under microscope (Nikon, Japan), which was proportional to cell proliferation.

Cell apoptosis assay

Apoptosis of HepG2 cells after kaempferol treatment and/or miR-21 mimic transfection were determined using Guava Nexin Assay Kit (Guava Technologies, Hayward, CA, USA) following the manufacturer’s instruction. Briefly, HepG2 cells were seeded, in triplicate, in 24-well plate (Thermo Fisher Scientific) with a density of 3 × 104 cells/well. After 50 μM kaempferol treatment for 24 h and/or miR-21 mimic transfection, cells were harvested, washed with phosphate-buffered saline (PBS), and stained with kit solution for 25 min at 37°C in the dark. Cell apoptosis was recorded using Guava EasyCyte flow cytometer (Guava Technologies). Data were analyzed using FCS Express software (De Novo Software, Los Angeles, CA, USA).

Cell migration and invasion assay

Migration of HepG2 cells was assessed using a modified two-chamber migration assay (BD Pharmingen, San Diego, CA, USA) with a pore size of 8 mm. After 50 μM kaempferol treatment and/or miR-21 mimic transfection, 1 × 103 HepG2 cells were suspended in 200 mL serum-free DMEM and seeded into top chamber. Complete DMEM (600 mL) was added into the lower chamber. After incubation for 48 h, cells were fixed with methanol (Beyotime Biotechnology, Shanghai, China) immediately. Non-traversed cells in top chamber were moved using cotton swab carefully and traversed cells in lower chamber were counted under microscope (Nikon, Japan). Cell migration (%) was calculated as follows: average traversed cells in treatment (transfection) group/average traversed cells in control group × 100%.

Cell invasion was conducted similarly with the cell migration assay except that the polycarbonate filter was pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) in line with the manufacturer’s instruction.

Quantitative reverse transcription polymerase chain reaction

The mRNA expressions of miR-21 and PTEN in HepG2 cells after relevant treatment or transfection were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Total RNA in HepG2 cells was extracted using TRIzol™ Plus RNA Purification Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cambridge, MA, USA). mirVana™ qRT-PCR miRNA Detection Kit (Invitrogen) was performed to detect the expression of miR-21 and the expression of U6 acted as the endogenous control. DyNAmo HS SYBR Green qPCR Kit (Thermo Fisher Scientific) was performed to detect the expression of PTEN and the expression of β-actin acted as the endogenous control. Data were quantified using 2−ΔΔCt method.[22]

Cell transfection

miR-21 mimic, miR-21 inhibitor, scramble, and negative control (NC) were all designed and synthesized by GenePharma (Shanghai, China). Sequences for miR-21 mimic were as follows: 5′-UAGCUUAUCAGACUGAUGUUGA-3′ (sense) and 5′-AACAUCAGUCUGAUAAGCUAUU-3′ (antisense). Sequence for miR-21 inhibitor was as follows: 5′-UCAACAUCAGUCUGAUAAGCUA-3′. Cell transfection was conducted using Lipofectamine 3000 (Invitrogen) in line with the manufacturer’s protocol. The stably transfected cells were selected using culture medium supplemented with 0.5 mg/mL G418 (Sigma–Aldrich). qRT-PCR was performed to assess the transfection efficiency.

Western blotting

After relevant treatment or transfection, total proteins in HepG2 cells were isolated using M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific). BCA Protein Assay Kit (Beyotime Biotechnology) was used to calculate the concentrations of proteins. Western blotting system was established using Bio-Rad Bis-Tris Gel System (Bio-Rad Laboratories, Hercules, CA, USA) in line with the manufacturer’s instruction. Proteins (45 μL) were electrophoresed in polyacrylamide gels and transferred onto nitrocellulose (NC; Millipore, Burlington, MA, USA) membranes, which were incubated with relevant antibodies. All primary antibodies were diluted in 1% bovine serum albumin (BSA; Beyotime Biotechnology) at a dilution of 1:1000. Anti-Cyclin D1 antibody (ab21699), anti-pro-caspase 3 antibody (ab90437), anti-cleaved-caspase 3 antibody (ab2302), anti-pro-caspase 9 antibody (ab32068), anti-cleaved-caspase 9 antibody (ab2324), anti-Bcl-2 antibody (ab59348), anti-Bax antibody (ab182733), anti-matrix metalloproteinase 2 (MMP-2) antibody (ab37150), anti-MMP-9 antibody (ab73734), anti-Vimentin antibody (ab137321), anti-t-phosphatidylinositol 3-kinase (PI3K) antibody (ab28356), anti-p-PI3K antibody (ab182651), anti-t-protein kinase 3 (AKT) antibody (ab8805), anti-p-AKT antibody (ab38449), anti-t-mechanistic target of rapamycin (mTOR) antibody (ab32028), anti-p-mTOR antibody (ab63552), anti-t-S6 K antibody (ab9366), anti-p-S6 K antibody (ab131459), and anti-β-actin antibody (ab8226) were all purchased from Abcam Biotechnology (Cambridge, MA, USA). After that, membranes were incubated with goat anti-rabbit (or anti-mouse) IgG H&L (HRP) (ab205718, ab205719; Abcam Biotechnology) for 1 h at room temperature and transferred into Bio-Rad ChemiDoc™ XRS system (Bio-Rad Laboratories), supplemented with 200 μL of Immobilon Western Chemiluminescent HRP Substrate (Bio-Rad Laboratories) on the surface of membranes. The intensities of the bands were quantified using Image Lab™ Software (Bio-Rad Laboratories).[23]

Statistical analysis

All experiments were repeated at least three times in our research. Results of multiple experiments were presented as the mean ± standard deviation (SD). GraphPad 6.0 Software (GraphPad, San Diego, CA, USA) was used to statistical analysis. The statistical comparisons (P-values) between two groups were calculated using Student’s t-test and P-values between more than three groups were calculated using one-way analysis of variance (ANOVA). P < 0.05 was considered to be a significant difference, and P < 0.01 and P < 0.001 were considered to be extremely significant difference.

Results

Kaempferol inhibits proliferation and induced apoptosis of HepG2 cells

Viability, proliferation, and apoptosis of HepG2 cells after kaempferol treatment were measured using CCK-8 assay, BrdU incorporation assay and Guava Nexin assay, respectively. Figure 1(b) shows that kaempferol inhibits the viability of HepG2 cells in a dose-dependent manner (P < 0.05, P < 0.01, or P < 0.001). Considering that the IC50 value is calculated as 48.26 μM, 50 μM is chosen for further experiments. Figure 1(c) displays that 50 μM kaempferol treatment significantly inhibits HepG2 cell proliferation (P < 0.01). The protein expression of Cyclin D1 in HepG2 cells after 50 μM kaempferol treatment is also decreased (Figure 1(d), P < 0.05). Figure 1(e) shows that the rate of apoptotic cells after 50 μM kaempferol treatment is remarkably increased (P < 0.001). Western blotting displays that kaempferol treatment up-regulates the expressions of cleaved-caspase 3, cleaved-caspase 9, and Bax, but down-regulates the expression of Bcl-2 in HepG2 cells (Figure 1(f)). The above findings suggest that kaempferol inhibits HepG2 cell proliferation but induces cell apoptosis.

Kaempferol suppresses HepG2 cell migration and invasion

Migration and invasion of HepG2 cells after 50 μM kaempferol treatment are assessed using two-chamber migration (or invasion) assay, respectively. As present in Figure 2(a) and (b), both the relative migration and the invasion of HepG2 cells decreased after kaempferol treatment (P < 0.01). Western blotting displays that the expressions of MMP-2, MMP-9, and Vimentin, which play critical roles in cancer cell migration and invasion,[24,25] all obviously decreased after kaempferol treatment (Figure 2(c) and (d), P < 0.05). These results indicate that kaempferol suppresses the migration and invasion of HepG2 cells.

Figure 2.

Kaempferol suppresses HepG2 cell migration and invasion. (a, b) Migration and invasion of HepG2 cells after 50 μM kaempferol treatment were detected using two-chamber migration (or invasion) assay, respectively. (c, d) Western blotting was conducted to analyze the expressions of MMP-2, MMP-9, and Vimentin in HepG2 cells after 50 μM kaempferol treatment.

MMP: matrix metalloproteinase.

*P < 0.05; **P < 0.01.

Kaempferol down-regulates the expression of miR-21 in HepG2 cells

The effect of kaempferol on miR-21 expression in HepG2 cells is detected using qRT-PCR. As displayed in Figure 3, the expression of miR-21 in HepG2 cells significantly decreases after 50 μM kaempferol treatment (P < 0.01). This result indicates that kaempferol down-regulates the expression of miR-21 in HepG2 cells and implies that miR-21 may be involved in the effects of kaempferol on HepG2 cell proliferation, apoptosis, migration, and invasion.

Figure 3.

Kaempferol reduces the expression of miR-21 in HepG2 cells. After 50 μM kaempferol treatment, the expression of miR-21 in HepG2 cells was determined using quantitative reverse transcription PCR (qRT-PCR).

miR-21: microRNA-21.

**P < 0.01.

Kaempferol inhibits proliferation and induced apoptosis of HepG2 cells by down-regulating miR-21

To analyze the roles of miR-21 in kaempferol-induced HepG2 cell proliferation inhibition and apoptosis enhancement, miR-21 mimic and miR-21 inhibitor are transfected into HepG2 cells, respectively. Figure 4(a) shows that the expression of miR-21 is significantly enhanced after miR-21 mimic transfection (P < 0.01) and dramatically reduced after miR-21 inhibitor transfection (P < 0.01). Figure 4(b) displays that miR-21 mimic transfection remarkably alleviates the kaempferol-induced HepG2 cell proliferation inhibition (P < 0.05). Compared to single kaempferol treatment group, the expression of Cyclin D1 in HepG2 cells is significantly increased in kaempferol treatment + miR-21 mimic transfection group (Figure 4(c), P < 0.01). Figure 4(d) shows that miR-21 mimic transfection obviously attenuates the kaempferol-induced HepG2 cell apoptosis (P < 0.01). Figure 4(e) displays that miR-21 mimic transfection alleviates the kaempferol-induced cleaved-caspase 3, cleaved-caspase 9, and Bax expressions increased, as well as Bcl-2 expression decreased. These findings suggest that miR-21 participates in the effects of kaempferol on HepG2 cells, and kaempferol inhibits HepG2 cell proliferation and induces cell apoptosis by down-regulating miR-21.

Figure 4.

Kaempferol inhibits proliferation and induced apoptosis of HepG2 cells by down-regulating miR-21. (a) After miR-21 mimic or miR-21 inhibitor transfection, the expression of miR-21 in HepG2 cells was detected using quantitative reverse transcription PCR (qRT-PCR). (b) Proliferation of HepG2 cells after 50 μM kaempferol treatment and/or miR-21 mimic transfection were measured using 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. (c) After 50 μM kaempferol treatment and/or miR-21 mimic transfection, the expressions of Cyclin D1 in HepG2 cells were assessed using western blotting. (d) Apoptosis of HepG2 cells after 50 μM kaempferol treatment and/or miR-21 mimic transfection were determined using Guava Nexin assay. (e) Western blotting was conducted to analyze the expressions of pro-caspase 3, cleaved-caspase 3, pro-caspase 9, cleaved-caspase 9, Bcl-2, and Bax in HepG2 cells after 50 μM kaempferol treatment and/or miR-21 mimic transfection.

miR-21: microRNA-21; NC: negative control.

*P < 0.05; **P < 0.01; ***P < 0.001.

Kaempferol suppressed HepG2 cell migration and invasion by down-regulating miR-21

Figure 5(a) and (b) displays that miR-21 mimic transfection remarkably alleviates the kaempferol-induced HepG2 cell migration and invasion inhibition (P < 0.05). Western blotting presents that the expressions of MMP-2, MMP-9, and Vimentin in HepG2 cells are increased after kaempferol treatment + miR-21 mimic transfection compared to single kaempferol treatment (Figure 5(c) and (d), P < 0.01). These above results further suggest that miR-21 is also involved in the kaempferol-induced HepG2 cell migration and invasion inhibition.

Figure 5.

Kaempferol suppresses HepG2 cell migration and invasion by down-regulating miR-21. (a, b) Relative migration and invasion of HepG2 cells after 50 μM kaempferol treatment and/or miR-21 mimic transfection were assessed using two-chamber migration (or invasion) assay, respectively. (c, d) Western blotting was used to analyze the expressions of MMP-2, MMP-9, and Vimentin in HepG2 cells after 50 μM kaempferol treatment and/or miR-21 mimic transfection.

miR-21: microRNA-21; MMP: matrix metalloproteinase; NC: negative control.

*P < 0.05; **P < 0.01.

miR-21 negatively regulates the expression of PTEN in HepG2 cells

qRT-PCR is performed to detect the mRNA expression of PTEN in HepG2 cells after miR-21 mimic or miR-21 inhibitor transfection. As displayed in Figure 6, the mRNA expression of PTEN is significantly reduced after miR-21 mimic transfection (P < 0.05) and remarkably enhanced after miR-21 inhibitor transfection (P < 0.01). These findings imply that PTEN may participate in the regulatory effects of miR-21 on kaempferol-induced HepG2 cell proliferation, migration and invasion inhibition, as well as apoptosis enhancement.

Figure 6.

miR-21 negatively regulates the expression of PTEN in HepG2 cells. Quantitative reverse transcription PCR (qRT-PCR) was performed to measure the expression of PTEN in HepG2 cells after miR-21 mimic or miR-21 inhibitor transfection.

miR-21: microRNA-21; PTEN: phosphatase and tensin homologue; NC: negative control.

*P < 0.05; **P < 0.01.

Kaempferol inactivates PI3K/AKT/mTOR signaling pathway in HepG2 cells by down-regulating miR-21

The effects of kaempferol treatment and/or miR-21 mimic transfection on PTEN expression and activation of PI3K/AKT/mTOR in HepG2 cells are measured using qRT-PCR and western blotting, respectively. Figure 7(a) shows that kaempferol treatment remarkably up-regulates the mRNA expression of PTEN in HepG2 cells (P < 0.01) and miR-21 mimic transfection obviously reverses the kaempferol-induced PTEN expression increase (P < 0.01). Figure 7(b) displays that kaempferol treatment down-regulates the expressions of p-PI3K, p-AKT, p-mTOR, and p-S6 K in HepG2 cells (P < 0.05 or P < 0.01) and miR-21 mimic transfection dramatically reverses the kaempferol-induced p-PI3K, p-AKT, p-mTOR, and p-S6 K expressions decrease (P < 0.01). These above findings imply that kaempferol inactivates PI3K/AKT/mTOR signaling pathway in HepG2 cells by down-regulating miR-21.

Figure 7.

Kaempferol inactivates PI3K/AKT/mTOR signaling pathway in HepG2 cells by down-regulating miR-21. (a) Relative expressions of PTEN in HepG2 cells after 50 μM kaempferol treatment and/or miR-21 mimic transfection were assessed using quantitative reverse transcription PCR (qRT-PCR). (b) Western blotting was conducted to analyze the expressions of t-PI3K, p-PI3K, t-AKT, p-AKT, t-mTOR, p-mTOR, t-S6 K, and p-S6 K in HepG2 cells after 50 μM kaempferol treatment and/or miR-21 mimic transfection.

miR-21: microRNA-21; PTEN: phosphatase and tensin homologue; NC: negative control; PI3K: phosphatidylinositol 3-kinase; AKT: protein kinase 3; mTOR: mechanistic target of rapamycin.

*P < 0.05; **P < 0.01.

Discussion

Liver cancer has become the third leading cause of cancer-related death worldwide.[2,26] This study revealed that kaempferol, a flavonoid compound, significantly inhibited liver cancer HepG2 cell proliferation, migration, and invasion, and induced cell apoptosis. Moreover, kaempferol remarkably down-regulated the expression of miR-21 in HepG2 cells and miR-21 was involved in the effects of kaempferol on HepG2 cell proliferation, migration, invasion, and apoptosis. Furthermore, miR-21 negatively regulated the expression of PTEN in HepG2 cells. Kaempferol inactivated PI3K/AKT/mTOR signaling pathway by down-regulating miR-21.

Natural medicines in cancer therapy have gained wide attention all over the world due to their safety, efficiency, and mini side-effects.[27] Epidemiological study demonstrated that there was a negative association between the consumption of foods containing kaempferol and the occurrences of cancers.[28] This research found that kaempferol dramatically suppressed liver cancer HepG2 cell viability, proliferation, migration, and invasion. The expressions of Cyclin D1, MMP-2, MMP-9, and Vimentin, which play important regulatory roles in promoting cell proliferation, migration, and invasion, respectively,[24,25,29] all significantly decreased after kaempferol treatment. Besides, kaempferol remarkably promoted HepG2 cell apoptosis. The expressions of cleaved-caspase 3, cleaved-caspase 9, and Bax, which exert pro-apoptotic roles in cancer cells,[30] increased and the expression of Bcl-2, which exerts anti-apoptotic role in cancer cells,[31] decreased after kaempferol treatment. Considering that inhibiting tumor cell proliferation and metastasis, as well as inducing tumor cell apoptosis, were most effective ways to cancer treatment, the findings of this research further verified the effective anti-tumor effects of kaempferol on liver cancer. Moreover, these findings were consistent with the previous study, which indicated that kaempferol significantly suppressed the proliferation of HepG2 cells and induced cell death.[14]

A previous study has demonstrated that microRNAs participate in the pathogenesis of multiple diseases, including cancers.[32] A variety of medicines can inhibit cancer cell proliferation and metastasis by modulating the expression of microRNA in cancer cells.[33] Previous research revealed that kaempferol reduced vascular smooth muscle cell migration through modulating BMP-mediated miR-21 expression.[21] This study found that kaempferol significantly down-regulated the miR-21 expression in HepG2 cells. In addition, overexpression of miR-21 obviously alleviated the effects of kaempferol on HepG2 cell proliferation, migration, invasion, and apoptosis. These findings indicated that miR-21 was also involved in the effects of kaempferol on liver cancer cells and implied that kaempferol exerted anti-tumor effects on liver cancer at least partially via down-regulating miR-21.

PTEN is an important and famous tumor suppressor, which is inactivated in multiple cancer cells.[34] Experimental researches have proved that PTEN is one of the direct target genes of miR-21.[35,36] This research found that the expression of PTEN significantly decreased after miR-21 overexpression and remarkably increased after miR-21 suppression in HepG2 cells. These findings were consistent with the previous study, which indicated that miR-21 modulated the expression of PTEN in human hepatocellular cancer cells.[20] Moreover, kaempferol remarkably enhanced the expression of PTEN in HepG2 cells. miR-21 overexpression notably reversed the kaempferol-induced PTEN expression increase. Considering the fact that PTEN played critical roles in suppressing cancer cell proliferation, migration, and invasion,[34] these results in our research implied that PTEN might participate in the regulatory effects of miR-21 on kaempferol-induced HepG2 cell proliferation, migration, and invasion inhibition, and apoptosis enhancement.

Kashafi et al.[37] demonstrated that kaempferol promoted human cervical cancer HeLa cell apoptosis through inhibiting PI3K/AKT pathway. Gwak et al.[38] indicated that silencing of miR-21 contributed to radio-sensitivity of malignant glioma cells by suppressing PI3K/AKT pathway. The activation of PI3K/AKT/mTOR signaling pathway participates in the occurrence of liver cancers.[39] Thus, this research further investigated the effects of kaempferol and miR-21 on PI3K/AKT/mTOR pathway in HepG2 cells. We found that kaempferol treatment significantly reduced the activation of PI3K/AKT/mTOR pathway in HepG2 cells. Overexpression of miR-21 dramatically reversed the effects of kaempferol on PI3K/AKT/mTOR pathway. These findings suggested that kaempferol inactivated PI3K/AKT/mTOR signaling pathway by down-regulating miR-21.

To sum up, this research verified that kaempferol inhibited proliferation, migration, and invasion of liver cancer HepG2 cells by down-regulation of miR-21 and up-regulation of PTEN, as well as inactivation of PI3K/AKT/mTOR signaling pathway. This study will be helpful for further understanding the anti-cancer effects of kaempferol on liver cancer and provides theoretical basis for deeply exploring the treatment of liver cancer using kaempferol.