Antitumor activity of a Rhodococcus sp. Lut0910 isolated from polluted soil

Abstract

The actinomycetes strain, lut0910, was isolated from polluted soil and identified as the Rhodococcus species with 99% similarity based on the sequence analysis of 16S recombinant DNA. The extract of this strain demonstrated in vivo and in vitro antitumor activity. The treatment of two human cancer cell lines, hepatocellular carcinoma HepG2 and cervical carcinoma Hela cells, with the lut0910 extract caused the delay in cell propagation in a dose-dependent manner with an IC₅₀ of 73.39 and 33.09 µg/mL, respectively. Also, the oral administration of lut0910 extract to the mice with a solid tumor resulted in the inhibition of tumor growth in comparison with a placebo group. The thymus and spleen indexes were significantly increased in mice groups treated with the lut0910 extract. The histopathological changes of the tumor tissues showed that there were massive necrotic areas in the tumor tissues after treatment with different doses of the lut0910 extract. Our result would provide a new way and potent source for development of new anticancer agent from the polluted environment.

Introduction

Cancer is a major health problem worldwide. One in four deaths in the United States is due to cancer. Asia accounts for 60% of the world population and half the global burden of this disease. The incidence of cancer cases is estimated to increase from 6.1 million in 2008 to 10.6 million in 2030, due to considerable growth in older population, improved lifestyle, and socioeconomic changes. Speaking of China, cancer is the leading cause of death in this country and is a major public health issue.^1,2

The cancer treatment is still a vital problem for health workers and pharmaceutical industry. More than 60% of the effective cancer chemotherapeutic agents used today are compounds identified from the soil microorganisms or synthesized compounds with modified properties. ^3,4 Many microbial metabolites are essential drugs for the cancer treatment. Their application for health needs started around 1940 with the discovery of actinomycin, and since then, many compounds with anticancer properties have been isolated from natural sources.³ So far, more than 23,000 bioactive secondary metabolites produced by microorganisms have been reported, and over 10,000 of them are produced by actinomycetes, representing 45% of all bioactive microbial metabolites discovered.⁵

Among actinomycetes, the Streptomyces genus has been widely studied over the last 60 years that resulted in the discovery of more than 7600 bioactive compounds including antitumor drugs.⁵ However, the rate of finding novel bioactive metabolite from Streptomyces species was recently decreased because of the nature of this well-studied subject and availability of screening technologies that were applied for several years worldwide to find such compounds.⁶

Rare actinobacteria are always referred to as strains that are difficult to isolate and might correspond to the unmatched source of new natural metabolites.⁷ In recent times, much attention has been given to isolate rare actinomycetes from diverse previously unexplored common as well as uncommon extreme environments.⁶ Many novel compounds discovered from the novel rare actinobacteria have been proven as potential new drugs in medical and pharmaceutical industries such as antibiotics, antimicrobials, antibacterials, anticancer, and antifungals.^{7 –9} Therefore, in this study, actinomycetes strain was isolated from the polluted soil. The extract from this strain was used to evaluate its antitumor activity in vitro and in vivo. This study would also provide a new way to find the antitumor activity compound from microorganisms in the polluted environment.

Materials and methods

Cell, chemicals, and reagents

Cell lines: human hepatoma cell line HepG2 and human cervical carcinoma Hela cells were kindly provided by Key Laboratory of Preclinical Study for New Drug of Gansu Province. Mouse H22 hepatocarcinoma cells were kindly supplied by Cancer Research Institute of Gansu Province. RPMI 1640 and fetal bovine serum (FBS) were purchased from GIBCOL company. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Rose Bengal, glucose, trypsin, beef extract, and peptone were purchased from Sigma Company. Dimethylsulfoxide (DMSO) and soluble starch were purchased from Beijing Tianwei Company. All other chemical regents were of analytical reagent grade and purchased from Kebaite Technology (Lanzhou, China).

Sample collection and strain isolation

The soil samples (approximately 50 g) were collected 5–10 cm below soil surface, in paper bags, from polluted soil in Jinchang District, Gansu Province, China. The soil samples were separately placed in beef extract peptone medium (5.0 g beef extract, 10.0 g peptone, 15 g agar, 5 g NaCl, 1000 mL distilled water, pH = 7.2), Rose Bengal medium (5 g peptone, 10 g glucose, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 15 g agar, 0.03 g Rose Bengal, 0.1 g chloramphenicol, 1000 mL distilled water, pH = 7.2), and Gause’s synthetic agar (20 g soluble starch, 1 g KNO₃, 0.5 g NaCl, 0.5 g K₂HPO₄·3H₂O, 5 g MgSO₄·7H₂O, 0.01 g FeSO₄·7H₂O, 1000 mL distilled water, pH = 7.2; 0.01% potassium dichromate was added to inhibit bacterial and fungal growth) and cultivated at 28°C ± 2°C for 24–96 h until a single colony appeared. Then, each colony was picked and stored for further analysis of their antitumor activity.

DNA extraction and identification of strain

Genomic DNA was extracted from high-activity strains Lut0910 using Ezup Column Genome DNA Extracting Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s protocol. The 16S recombinant DNA (rDNA) from the extracted DNA was amplified by polymerase chain reaction (PCR) using primer 27f (5′-AGAGTT TGATCMTGGCTCAG) and primer 1492r (5′-GGTTACCTTGTTACGA CTT).¹⁰

PCR was carried out in 50 µL reaction mixtures containing 5 µL 10× PCR Buffer, 4 µL 2.5 mmol/L dNTPs, 1 µL 5 µmol/L forward primer, 1 µL 5 µmol/L reverse primer, 0.5 µL Taq enzyme, 1 µL template DNA and 50 µL of double-distilled H₂O. The conditions used for thermal cycling were as follows: denaturation at 94°C for 5 min, followed by 32 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and an extension at 72°C for 50 s. At the end of the cycles, the reaction mixture was kept at 72°C for 10 min and then cooled to 4°C. PCR products were detected with 1% gel electrophoresis and analyzed by Sangon Biotech.

The 16S rDNA gene sequences were BLAST searched against the GenBank database (http://www.ncbi.nlm.nih.gov/). The sequences determined and reference sequences downloaded from GenBank were aligned using multiple-sequence alignment software CLUSTAL X version 1.81 Phylogenetic.

Preparation of fermentation broth extract

The isolated microorganisms were grown in 500 mL liquid Rose Bengal medium at 30°C with shaking at 180 r/min for 48 h. Centrifugation at 7100 g/min for 10 min was carried out, and the supernatant of the bacterial suspension was deproteinized by Sevage method with shaking sample:chloroform:butyl alcohol in the volume of 25:5:1 (v/v/v) in the container for 10 min and then was allowed to stand for 12 h at 4°C and centrifuged at 7100 g/min for 30 min. This procedure was repeated three times until the proteins were not detected in the supernatant using the Coomassie Brilliant Blue staining. The supernatant was concentrated to 1/10 volume in a rotary evaporator under reduced pressure, and then, the extract was dried at 60°C and stored at 4°C for further use.

Antitumor activity assay

Evaluation of cell growth inhibition in vitro

Tumor cell lines and culture condition

Human cancer cell lines (Hela cells) and human hepatocellular carcinoma HepG2 cells were maintained in RPMI 1640 medium supplemented with 2 mmol/L l-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mmol/L (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1.0 mmol/L sodium pyruvate, 10% FBS, 100 U/mL of penicillin, and 100 µg/mL streptomycin at 37°C with 5% CO₂.

In vitro experiment

Antitumor activity of extract was conducted using MTT assay.^11,12 The human hepatocellular carcinoma HepG2 and cervical carcinoma Hela cells were collected at the stage of logarithmic growth using 0.25% trypsin. More than 98% of cells were alive according to Trypan blue staining. Cells were seeded in 96-well culture plates at an optimal density of 5 × 10³ cells per well for 24 h at 37°C with 5% CO₂ and the extract was added. The final concentrations of the extracts were 5 (10), 10 (12.5), 20 (25), 50, and 100 µg/mL. A blank control and negative control (without the extracts) were also included. Then, 50 µL of 5 mg/mL MTT solution was added per well at 48 h post-transfection. The medium was removed by centrifugation after 4 h of incubation at 37°C in 5% CO₂ incubator, and the blue formazan product converted from MTT was dissolved by the addition of 150 µL DMSO per well. The plates were vibrated at room temperature for 10 min, and then, the spectrophotometric absorbance was measured at 570 nm. All assays were performed in triplicate. Growth inhibition rate (IR) was calculated by the following formula

$$\mathrm{IR}=\frac{\mathrm{ODcon}.-\mathrm{ODtreated}}{\mathrm{ODcon}.}\times 100\%$$

where ODcon and ODtreated are ODs of control and treated wells, respectively.¹⁰

Evaluation of the antitumor activities in vivo

Cell and animals

Mouse H22 hepatocarcinoma cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal calf serum (FCS), 2.05 mM l-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin at 37°C in a humidified 5% CO₂ atmosphere.

A total of 40 BALB/c mice weighing (20 ± 2) g were obtained from the Lanzhou University Animal Center. According to the requirement of the National Act on the Use of Experimental Animal (People’s Republic of China), the protocols of animal experiments were approved by the Animal Ethics Committee of Lanzhou University of Technology. All animals were housed at room temperature 25°C ± 2°C and 12-h light–dark cycle. The animals were allowed free access to food (standard laboratory animal food) and water. After 7 days of acclimatization, all animals were randomly divided into different groups (n = 10) and each mice was used for only one experimental condition.

In vivo antitumor experiment

To establish murine solid tumors H22 transplanted model, H22 hepatocarcinoma cells were transferred into the abdomen cavity of Kunming mouse. The ascites were taken from the mouse and diluted with Hanks solution to 1 × 10⁷ cells/mL. Then, the corresponding ascites tumor cells (1 × 10⁷ mL/per mouse/0.15 mL) were subcutaneously injected into the right axillary region of the BALB/c mice in all groups. A total of 40 BALB/c mice were divided randomly into four groups 72 h after inoculation: NS control group (model control, 0.9% normal saline); 20 mg/kg cyclophosphamide (CTX group), and lut0910 low-(normal-) and high-dose (100 and 200 mg/kg, respectively) groups (n = 10).¹³ The BABL/c mice inoculated with H22 cells were orally administrated with corresponding doses of extracts one time daily for 10 days in a volume of 0.5 mL. The mice in model control group received an equal volume of normal saline. On the 11th day, the surviving mice in each group were sacrificed by cervical dislocation, and the body weight of mice was measured. Then, the tumors were immediately excised and weighed, and the tumor IR was calculated using the following formula

$$\mathrm{Tumor\; inhibitory\; rate}\left(\mathrm{\%}\right)=\left[\left(\mathrm{C}-\mathrm{T}\right)/\mathrm{C}\right]\times \mathrm{100}$$

where C is the average tumor weight of the model control group and T is the average tumor weight of the treated groups.

At the same time, the spleen and thymus were also removed and weighed for the calculation of organ indexes. The thymus and spleen indexes were expressed as the thymus and spleen weight (mg) relative to the body weight (g). The index of the thymus and spleen was calculated by the following equation^14,15

$$\mathrm{The\; indexes}\left(\mathrm{mg}/\mathrm{g}\right)=\frac{\mathrm{Thymus\; or\; spleen\; weight}\left(\mathrm{mg}\right)}{\mathrm{Body\; weight}\left(\mathrm{g}\right)}$$

Histopathological examinations analysis

The tumor of mice was collected for pathological sectioning with hematoxylin and eosin (H&E) staining according to Peng et al.¹⁶ The tumor tissue from the mice was fixed in 10% buffered formalin saline, embedded into paraffin, and then cut into 5-µm-thick sections. The sections were fixed and stained with H&E. The histopathological changes in tumor tissues were examined under Motic E220 microscope (Xiamen, China)¹⁶

Data analysis

All data were expressed as mean ± SD and analyzed by single-factor analysis of variance and least significant difference. Data were analyzed by one-way analysis of variance followed by Duncan’s multiple range test when the t-test was significant using the SPSS17.0 software. Statistically significant differences between control and experimental groups (p < 0.05) are indicated.

Results

Screening and isolation of lut0910 microorganism

The soil samples from polluted soil (Jinchang District, Gansu Province) were inoculated on the various medium, and 43 morphologically different colonies were found to grow well. With MTT assay, a red-colored colony was separated from other colonies on Rose Bengal medium (Figure 1(a)). The colony grew fast on Rose Bengal medium, had an ellipsoidal or spherical shape, and was smooth and wet (Figure 1(b)).

Figure 1.

Morphological characteristics and a phylogenetic tree of Rhodococcus sp. lut0910. (a) Colony of Rhodococcus sp. lut0910 pointed out with an arrow on the agar-based Rose Bengal seeded with a soil sample. (b) Colony of Rhodococcus sp. lut0910 on agar-based Rose Bengal medium. (c) Phylogenetic tree of Rhodococcus species.

[Figure omitted. See PDF]

A partial sequence of the 16S rDNA gene (1481 bp) was identified and deposited in GenBank at NCBI (Accession No. HQ228155.1). BLAST analysis revealed that lut0910 belonged to the Rhodotorula sp. genus. A phylogenetic tree was constructed using maximum-parsimony and neighbor-joining method with MEGA 5.0 software (Figure 1(c)). The highest 16S ribosomal RNA (rRNA) sequence similarities between the isolated and type strains of recognized species in the databases were 99%.

Cell growth inhibition of the lut0910 extract in vitro

MTT is a tetrazolium salt that can be reduced in the mitochondria of living cells and transformed into water-insoluble purple formazan. MTT reduction is widely used for the measurement of cell proliferation and viability.^17,18 In this study, we used MTT assay to investigate the effect of the lut0910 extract on the proliferation of two human cancer cell lines, hepatocellular carcinoma HepG2 and cervical carcinoma Hela cells. The cytotoxic effect of the lut0910 extract on cancer cell lines is shown in Figure 2. The tumor cell viability was dose-dependent on the lut0910 extract (Figure 2(a) and (b)) with the IC₅₀ values equal to 33.0851 ± 2.1564 µg/mL for HepG2 cells and 73.3963 ± 3.0876 µg/mL for Hela cells. Compared with the untreated controls, significant inhibition of the HepG2 and Hela cells growth was observed under treatment with 25–100 µg/mL concentrations of the lut0910 extract (p < 0.05). Moreover, the effects of the lut0910 extract on cell modality were observed by the inverted microscope for 24 h. The analysis of cell modality provided results consistent with MTT assays. As shown in Figure 2(c) and (e), the untreated HepG2 and Hela cells grew fast and displayed a normal shape, indicating a normal condition for these cells. However, following treatment with the lut0910 extract, the HepG2 and Hela cells exhibited a growth rate inhibition. Shrinkage, irregular enlargement, and vacuole on cell surface were distinguished in the lut0910-treated group when compared with controls; damaged cells or dead cells with evident morphological abnormalities were also found (Figure 2(d) and (f)).

Figure 2.

Cytotoxic activity of the lut0910 extracts against human cervical carcinoma cell Hela (a) and hepatocellular carcinoma cell HepG2 (b). Cell modality of Hela (c) and HepG2 (d) cells untreated, and cell modality of Hela (e) and HepG2 (f) cells treated with the lut0910 extracts (&,#p < 0.05, compared with a negative group).

[Figure omitted. See PDF]

Antitumor activity of the lut0910 extract in vivo

To investigate whether Lut0910 has an inhibitory effect on tumor growth in vivo, BALB/c mice with the H22-generated tumor were administered orally with lut0910 extracts (100 and 200 mg/kg/d), TLX, and vehicle (normal saline) for 10 days after H22 cancerous cell implantation. As shown in Figure 3(a), the results obviously demonstrated that the administration of the lut0910 extract could significantly decrease tumor growth and prevent tumor diffusion. In addition, the administration of the lut0910 extract led to significant decrease in the tumor volume and the spreading of the tumor to surrounding tissues. The tumor weights were dramatically decreased in mice administered with the lut0910 extract when compared it with the model control group (Figure 3(b), p < 0.05). The lut0910-treated groups showed the significantly inhibited growth of H22-induced tumors. H22-induced tumors were inhibited by 47.82% and 38.05% at the dose of 100 and 200 mg/kg body weight, respectively. Moreover, compared with the CTX control group, there was no significant difference in the reduction of tumor weights of low- and high-lut0910-treatment group (p > 0.05). The results indicated that the lut0910 extract and cyclophosphamide have similar antitumor effects on the H22-generated tumor.

Figure 3.

Effect of the lut0910 extract on H22-induced tumor growth. (a) Photographs of tumors obtained from normal control, CTX control, high-dose, and low-dose groups. (b) Tumor weight for two control and two tested groups. (c) Histological analysis of tumor tissues of the control and tested groups.

[Figure omitted. See PDF]

Effect of the lut0910 extract on the spleen and thymus

Thymus and spleen play important roles in the immune system by resisting foreign invaders. Their weight gains can indirectly imply the functions of immune systems. To evaluate whether the administration of the lut0910 extract results in any side effects on the immune system, the thymus and spleen indexes were determined at the end of the study (Table 1). The spleen and thymus indexes for mice in the CTX-treated group were significantly lower than those in the model control group (p < 0.05). Moreover, spleen index was significantly increased in the lut0910-treated groups (p < 0.05) than in the model control group. These data suggested that the administration of the lut0910 extract had a beneficial effect on immune organs of mice with H22-induced tumor.

Table 1.

Effects of direct treatment with the lut0910 extract at the dosage of 100 and 200 mg/kg on thymus index and spleen index of H22-induced tumor in mice (n = 10).

Treatment group	Dose (mg/kg)	Spleen index (mg/g)	Thymus index (mg/g)
NS control	–	10.8256 ± 2.6865*	2.1166 ± 0.2551*
CTX	20	9.7563 ± 2.2330#	1.7936 ± 0.8879#
Low doses	100	14.6570 ± 3.8859*#	1.9254 ± 0.1724*
High doses	200	15.2037 ± 5.4363*#	2.2978 ± 0.4708*

NS Control: model control, in which mice received 0.9% normal saline; CTX: positive control, in which mice received 20 mg/kg CTX; high doses and low doses are groups of mice that received 100 and 200 mg/kg the lut0910 extract, respectively.

Values are represented as mean ± SD.

*

p < 0.05, compared with the CTX group.

#

p < 0.05, compared with the NS control group.

Histopathology of tumors

H&E staining is one of the most common methods used in histopathology.¹⁹ The histopathological changes in the tumor tissues are shown in Figure 3(c). In contrast to the vigorous growth of the tumor cells in the normal saline group (Figure 3(c)), there were massive necrotic areas in the tumor tissues, revealed that spreading of tumor cells was decentralized and accompanied with many vacant regions caused by phagocytosis. Muscular tissues encapsulated groups of tumor cells after treatment with different doses of the lut0910 extract. The results indicated that application of the lut0910 extract could effectively restrain tumor growth in vivo.

Discussion

Actinomycetales are non-motile Gram-positive bacteria that live in a broad range of environments, including soil, water, and eukaryotic cells. This group comprises some of the most important organisms known to humankind and are known to possess a variety of interesting and useful metabolic capacities such as antitumor property.²⁰ The treatment and prevention of cancer are particularly important due to the rapidly increasing cancer mortality rate worldwide.¹⁶ In this study, we reported the isolation and characterization of a soil actinomycete from contaminated soil enrichments and the evaluation of its antitumor property. Our results showed that cytotoxicity of the lut0910 extract on human HepG2 and Hela cells was dose-dependent. Interestingly, the lut0910 extract had a better cytotoxicity activity on HepG2 cells with a lower IC₅₀ when compared with Hela cells.

Previous studies have shown that the ascitic-type liver cancer H22 can grow in many mouse strains with high invasiveness and metastasis. The facility and stability of the H22-induced tumor model made it one of the most popular tumor models in research.²¹ Since the lut0910 extract showed antiproliferation activities against HepG2 cancer cell line, we further used an H22-xenografted mouse model to evaluate the antitumor activity of the lut0910 extract in vivo. Our results revealed that both the lut0910 extract and TLX could significantly inhibit the tumor growth in H22-generated cancer model mice. Both of them caused tumor regression compared to the control group, as shown in Figure 3.

Rose Bengal (4,5,6,7-tetrachloro-2,4,5,7-tetraiodofluorescein disodium salt) is a xanthene dye produced by combining halogens with fluorescein that was patented in 1882.²² Smith and Dawson²³ first described the effect of the bacteriostatic action of Rose Bengal in media used for soil fungi. Although the successful application of Rose Bengal for the isolation of fungi and yeast has been demonstrated repeatedly,^24,25 Ottow²⁴ confirmed that this dye could also be used as a selective reagent for the isolation of actinomycetes from natural sources. In this study, a Rose Bengal medium was used for the isolation of microorganisms from the contaminated natural environment, and the resulting active strain was identified as Rhodococcus sp.

The genus Rhodococcus was revived and redefined in 1977. It did not belong to the established genera Nocardia, Corynebacterium, and Mycobacterium. Rhodococci are described as aerobic, Gram-positive, non-motile, mycolate-containing nocardioform actinomycetes.²⁶ Rhodococci have an environmental and biotechnological importance because of their broad catabolic diversity and array of unique enzymatic capabilities.^26,27 Applications of Rhodococci include bioactive steroid production, fossil fuel biodesulfurization, and the production of acrylamide and acrylic acid, the most commercially successful application of a microbial biocatalysis.²⁰ However, there are few reports about pharmaceutical bioactivity of metabolites of the Rhodococcus strains. Here, the antitumor activity of the extract from the Rhodococcus sp. lut0910 was found for the first time in vitro and in vivo.

Although studies on the antitumor metabolic activity of Rhodococcus sp. are rare in the literature, the microorganism is widely used in the degradation of toxic compounds; thus, it is also possible to find the metabolite or synthetic product of the strain with antitumor activity. For the first time, this study confirmed that Rhodococcus sp. and some other strains with specific metabolic pathways may also exist in the polluted environment and produce compounds with antitumor activity. This study also provides a new approach for finding new candidate compounds from the polluted environment.

Some studies reported that Rose Bengal was cytotoxic as it caused lysozyme-induced and apoptotic cell death in various human and murine malignancies.^22,28,29 To confirm that the cytotoxic activity of the lut0910 extract obtained in this study is not derived from Rose Bengal, the lut0910 strain was grown in the medium with or without Rose Bengal. The antitumor activities of two lut0910 extracts produced from the bacteria grown in the medium with or without Rose Bengal were tested on HepG2 cells. The results were similar demonstrating that the antitumor activity of the lut0910 extracts was not associated with Rose Bengal (Figure 4(a)). Moreover, the antitumor activity of the lut0910 extracts evaluated after treatment at 4°C and 70°C for different times did not differ significantly, indicating that the active component of extraction had a high thermal stability (Figure 4(a)). In addition, the antitumor activity of the lut0910 extracts prepared with different solvents including ethyl acetate, butanol, and water was also evaluated, and the result showed that the aqueous extract had higher antitumor activity, suggesting that the active ingredient is mainly in the aqueous phase (Figure 4(a)). As shown in the antitumor activity time profile (Figure 4(b)), the antitumor activity of the lut0910 extracts gradually increased with the growth of the biomass. The antitumor activity was not elevated after the growth of the cells to the stationary phase, indicating that the antitumor active component may be the primary metabolite of the strain. These results will also provide the possibility for further isolation of the active compound from the fermentation broth.

Figure 4.

Evaluation of antitumor activity of different types of extracts prepared from (a) Rhodococcus sp. lut0910 strain and (b) its time–antitumor curve.

[Figure omitted. See PDF]

Conclusion

Although Rhodococcus sp. was used as an important resource for industrial biocatalysts, for example, for the large-scale production of acrylamide and acrylic acid and bioactive steroid compounds, the antitumor compounds that can be produced by this bacteria were not characterized. We demonstrate for the first time that Rhodococcus sp produces a compound that has antiproliferative activity in vivo and in vitro and could be an attractive candidate for anticancer therapy. This study also provided a new way to find the compounds from the polluted environment.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the program of National Natural Science Foundation of China (Grant No. 31360379).