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ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1 

 

CTMJ | traditionalmedicinejournals.com  

 

 

Impact of NLRP3 inhibition and micro-ribonucleic acid-22 up-

regulation on malignant melanoma cell survival 
 

D.VenkataramanDra 1, Dr.D.Suryadevarakonda 2, DivyaAIia Sahithi 3, 
Assistant professor 1,2,3, 

Department of Pharmacy, 
Samskruti College of Pharmacy, 

Kondapur (V), Ghatkesar (M) Medchal Dist, Telangana, India. 

 

 
 

Abstract 

The goal of this study is to identify the mechanisms by which the up-regulation of miR-22 targeting NLRP3 alters 

the proliferation and invasiveness of malignant melanoma cells and the effects on these metrics. Amelomas grown in 

mice Thawing, sub-cultivation, and transfection were performed on B16 cells. various transfections were used to 

assign cells to various groups (A1–A5). For example, A1 cells were transfected with a miR-22 mimic 

overexpression, A2 cells with a miR-22 inhibitor, A3 cells with a miR-22 mimic+siNLRP3 transfection, A4 cells with 

a miR-22 mimic NC sequence transfection, and A5 cells without transfection. The results showed that miR-22 

expression was increased in the A1 group compared to the A2, A3, A4, and A5 groups, but decreased in the A3, A4, 

and A5 groups. A1 had considerably lower levels of NLRP3 mRNA and protein compared to other groups, but A2 

had significantly higher levels compared to A3, A4, and A5 (p < 0.05). In comparison to groups A3, A4, and A5, the 

A2 group showed a considerable increase in cell proliferation and colony formation rate, while groups A1 showed a 

marked decrease. Reducing the incidence and development of malignant melanoma is achieved by targeting NLRP3 

suppression by up-regulating miR-22 expression level. This greatly decreases the proliferation, invasiveness, and 

matrix metalloproteinase levels of melanoma B16 cells. Insightful reference data for gene-level therapy of malignant 

melanoma is provided by the findings. Macular melanoma, micro-ribonucleic acid-22, NLRP3 inflammasome, cell 

invasion capacity, cell transfection  

 

 

 

 INTRODUCTION  
 

Malignant melanoma refers to cancerous 

transformation of the corresponding pigmented 

nevus. Due to the morphological changes in nevus 

cells, the tumor formed is called melanoma [1,2]. The 

etiology of melanoma is very complex. The most 

common risk parameters are environmental factors 

(sunlight exposure and chemical stimulation), genetic 

factors and immunological factors [3]. Melanoma is a 

highly malignant tumor that occurs mostly in the 

skin, but also in the mucous membranes and internal 

organs, and it accounts for about 3% of all tumors 

[4,5]. There has been a rise in fatality associated with 

this tumor in the past 12 years. 

 

Studies show that there are about 150,000 new 

melanoma cases and about 50,000 melanoma-related 

deaths worldwide every year [6-8]. Melanoma is very 

malignant, and it is liable to blood and lymphatic 

metastasis, and it has a high mortality. Timely 

surgical resection when there is no metastasis may 

prolong the survival time of patients by 3 - 5 years. 

Once malignant melanoma undergoes distant 

metastasis to multiple organs, it may likely lead to 

organ failure and death, and the prognosis is poor 

[9,10]. Therefore, early diagnosis and timely and 

reasonable treatment are essential.  

 

Surgery remains the primary and definitive treatment 

for early-stage melanoma, but it is rarely curative for 

advanced-stage melanoma [11]. In recent years, the 

applications of immuno- and targeted treatments have 

prolonged patients' lives and revolutionized the 

management of this neoplasm [12]. Unfortunately, 

many subjects develop resistance to targeted 

medications after several months, and studies suggest 

that micro-ribonucleic acid plays a key role in the 

development of drug insensitivity [13,14]. The 

miRNAs are a class of evolutionarily conserved, non-



 Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1 

 

CTMJ | traditionalmedicinejournals.com  

 

coding small ribonucleic acids generally between 21 - 

23 nucleotides in length, and they regulate gene 

expression at the translational level [15]. There are as 

many as 1000 different miRNAs in vertebrate 

genomes which regulate at least 30% of gene 

expressions, and they are highly conserved, tissue-

specific and time-sequential. The nucleotide-binding 

oligomerization domain, leucine-rich repeat and 

pyrin domain-containing 3 (NLRP3) inflammasome 

is a multi-protein structure that exists in cells in vivo, 

and it is a crucial part of innate immunity which 

regulates the activation of caspase-1 and promotes 

the release of pro-inflammatory factors, i.e., 

interleukins IL-1β/IL-18 [16-18]. Accumulated 

evidence suggests that the inflammasome plays a 

central role in regulating tumorigenesis, growth, 

invasion, and metastasis. It has been shown that IL-

18, the effector product of the NLRP3 

inflammasome, maintains the epithelial homeostasis 

of colons and plays a protective role in colitis-

associated colon cancer [19]. Some studies have also 

shown that NLRP3 enhanced carcinogenesis by 

affecting host immune response, cancer cell 

multiplication and microenvironment of cancer 

[20,21]. At present, the regulatory effect of miR-

22/NLRP3 axis on the biological function of tumor 

cells in colon, ovarian, gastric, and other malignant 

tumors, have been demonstrated. However, there is 

limited research on the relationship between miR-

22/NLRP3 and malignant melanoma.  

 

The mechanism that reveals how miR-22 targeting 

NLRP3 inhibits the expression of malignant 

melanoma is still not clear. Therefore, in this study, 

mouse-derived melanoma cells B16 cells were 

subjected to thawing, sub-culture, and transfection. 

Based on what was transfected, the cells were divided 

into 5 groups: A1 (overexpressed miR-22 mimic) A2 

group (miR-22 inhibitor), A3 group (transfected with 

miR-22 mimic+siNLRP3), A4 (miR-22 mimic NC 

sequence transfection), and A5 group (without 

transfection). The five groups of cells were compared 

with respect to miR-22 messenger ribonucleic acid 

(mRNA) expression, NLRP3 mRNA and protein 

expressions, proliferation potential, clone formation 

rate, invasiveness, and levels of inflammatory factors, 

amongst other parameters. Arising therefrom, the 

effect of NLRP3 inhibition-targeted miR-22 up-

regulation on proliferation and invasiveness of tumor 

cells was evaluated, and the related mechanism was 

also assessed.  

 

EXPERIMENTAL  

 

Melanoma cells  
 

Mouse-derived melanoma cells B16 cells purchased 

from Shanghai Guandao Bio-engineering Co. Ltd 

were used as experimental cells. The B16 cells were 

cultured in Roswell Park Memorial Institute (RPMI) 

1640 medium (modified with ATCC) tinged with 10 

% FBS. Thereafter, the cells were kept in liquid 

nitrogen.  

 

Cell thawing  

 

Centrifuge tubes, disposable pipettes, RPMI 1640 

medium and phosphate buffer saline (PBS) were 

sterilized for 20 min on an ultra-clean bench. Then, 

the B16 cells were removed from liquid nitrogen and 

thawed for 60 sec in a water bath preheated to 40 ℃. 

The cells were pipetted into a 15-mL centrifuge tube, 

and 6 mL of PBS solution was added, followed by 

centrifugation at 1300 rpm for 6 min. The supernatant 

was discarded, and the sediment was suspended in 6 

mL of RPMI 1640 medium. After thorough mixing, 

the cells were cultured in a cell culture dish in the 

presence of 5 % carbon dioxide (CO2) at 37 ℃. The 

medium was changed after 24 h, and sub-culture was 

performed until the cell growth density reached about 

98 %.  

 

Cell subculture  

 

Following a 20-min UV sterilization of a cleaned 

bench, the culture medium, trypsin, and PBS were 

pre-heated. The PBS in cell culture dish was replaced 

with 1.5 mL trypsin, and following mixing, the cells 

were left at 25 ℃ for about 3 min, after which cell 

morphology was examined under a high-definition 

microscope. When the cells became rounded, 2 mL 

of RPMI 1640 medium was added, and the cells were 

centrifuged at 1300 rpm for 6 min. The supernatant 

was replaced with 6 mL PBS solution, and the cells 

were centrifuged again at 1300 rpm for 6 min, and 

the supernatant was again replaced with RPMI 1640 

medium. The re-suspended cells were put into 5 new 

culture dishes and cultured at 37 ℃ in a 5 % CO2 

atmosphere.  

 

Cell transfection  

 

Before cell transfection, the B16 cells were seeded at 

concentrations of 105 to 106 cells/well in six-well 

plates. When 80 % confluence was attained, miR-22 

mimic was transfected into the cells. The transfection 

was carried out in line with the steps indicated in the 

transfection kit (Guangzhou Ribo Bio Company 

Ltd.). Five groups of cells were used (A1 - A5). In 

A1, transfection with miR-22 mimic overexpression 

was done. Transfection in A2 group of cells was done 

with miR-22 inhibitor. In A3 group, transfection was 



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with miR-22 mimic+siNLRP3. In A4 group, 

transfection was with miR-22 mimic NC sequence, 

while cells in A5 group were not transfected.  

 

Determination of cell proliferation  

 

The CCK-8 method was employed for determination 

of cell multiplication. Cells in log stage of growth 

were 0.25 %-trypsinized for 4 min, after which the 

cell morphology was examined under a high-

definition microscope. When the cells became 

rounded, addition of RPMI 1640 medium was 

followed by 6-min cell centrifugation at 1300 rpm, 

re-suspension in RPMI 1640 medium, and counting 

under a microscope using a counting plate. For this 

purpose, a drop of cell culture medium was placed on 

the counting plate, after which the cells were counted 

under a high-definition microscope, and the cell 

concentration was calculated. Then, cell seeding 

(density = 1 × 106 cells/mL) in a 96-well plate was 

followed by culturing in 80 μL of RPMI 1640 

medium for 24 h. After addition of 15 μL CCK-8 

reagent, the cells were incubated at 37 ℃ for 2 h, and 

the absorbance of each well was read at 520 nm.  

 

Cell clone formation experiment  
 

High-definition microscopy was used for 

morphological examination of B16 cells after cell 

trypsinization as before. After the cells became 

rounded, RPMI 1640 medium was introduced, after 

which the cells were centrifuged for 6 min at 1300 

rpm. Thereafter, re-suspension of the cells in RPMI 

1640 medium was done. Then, the cells were counted 

under a microscope using a counting plate to get the 

number of cell clones (NC). Next, 500 cells were 

placed in a culture dish and cultured for 1 day. The 

cultured cells were fixed with 4 % paraformaldehyde 

for 4 h, stained with crystal violet for 20 min, dried 

and counted under a high-definition microscope. The 

cell clone formation rate (CR) was calculated as 

indicated in Eq 1.  

 

CR = (NC/NI)100 ………………. (1)  

where NI is the number of cells inoculated  

 

Cell scratch test  

When logarithmic growth stage cells grew to 98 % 

confluence, three parallel lines were drawn on the 

bottom of the plate with a pipette tip. This was 

followed by culturing in serum-free RPMI 1640 and 

photographing under a fluorescence microscope on 

days 1, 3 and 5. The degree of cell fusion was 

calculated using Image J software.  

 

Cell invasion assay  

 

Logarithmic growth phase cells were inoculated in 

Tanswell upper chamber and cultured in serum-free 

RPMI 1640. Then, addition of RPMI 1640 medium 

having 10 % FBS was done to the 24-well plate 

underneath. Following 24-h culturing, 4 % 

paraformaldehyde fixation was done, and the cells 

were crystal violet-stained for 15 min. After drying, 

the cells were counted under a high-definition 

microscope.  

 

Evaluation of parameters/indices  
 

Real-time quantitative polymerase chain reaction 

(qRT-PCR) was used to determine the expression 

levels of miR-22, NLRP3, caspase-1 mRNA, and 

matrix metalloproteinases (MMP2 and MMP9). 

Western blot assay was used to measure the protein 

expression levels of NLRP3, caspase-3, and matrix 

metalloproteinases (MMP2 and MMP9), while levels 

of inflammatory factors (IL-6, IL-8, IL-10, and IL-

1β) were measured with ELISA.  

 

Statistical analysis  
 

The SPSS 19.0 software was applied for data 

processing. Measurement data are presented as mean 

± standard deviation (SD), and enumeration data are 

presented as %. Pairwise comparisons were 

performed using one-way ANOVA. Differences were 

assumed to be statistically significant at p < 0.05.  

 

RESULTS  

 

Expression levels of miR-22 in cells in the 5 groups  
Figure 1 a show miR-22 levels in the five groups of 

cells. There was a significant up-regulation of miR-

22 in A1 than in A2, A3, A4, and A5, but miR-22 

was significantly lower in A2 than in A3 - A5 (p < 

0.05). However, miR-22 level was comparable in 

groups A3, A4, and A5 (p > 0.05). Figure 1 b is the 

miR-22 PCR band map showing that A1 had the 

highest brightness, while A2 band was the lowest. 

Band brightness was similar in A3, A4, and A5 

groups. 



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Figure 1: Expression levels of miR-22 in the 5 

groups of cells. (a) miR-22 expression levels; (b) 

miR-22 PCR band map. *P < 0.05, vs. A1; #p < 0.05, 

vs. A2  

 

MRNA and protein levels of NLRP3 in the five 

groups  

 

Figures 2 a and b show the protein and mRNA levels 

of NLRP3, respectively, in all 5 groups, with A1 cells 

having significantly lower levels than A2, A3, A4, 

and A5 cells. However, NLRP3 mRNA and protein 

levels were highest in A2 (p < 0.05). Pairwise 

differences in mRNA and protein expressions of 

NLRP3 amongst A3, A4, and A5 groups were not 

significantly different. Band maps of PCR and 

western blot of NLRP3 are presented in Figures 2 c 

and d, respectively. The brightness of the bands of 

cells was highest in A1 group, while A2 group had 

the darkest band. The bands of cells in A3, A4, and 

A5 groups showed similar brightness. 

 

 

 
 

Figure 2: NLRP3 protein and mRNA levels in cells 

in the 5 groups. The NLRP3 mRNA expression, 

NLRP3 protein expression, PCR band diagram, and 

western blot band diagram are depicted in a, b, c, and 

d, respectively. *P < 0.05, vs. A1 group; #P < 0.05, 

vs. A2 

 

 

 
 

Figure 3: Comparison of cell proliferation and clone 

formation rate among the 5 groups. a, b, and c are 

cell proliferation, clone formation rate, and cell clone 

formation rate, respectively. *P < 0.05, vs A1 group; 

#p < 0.05, vs A2 group  

 

Cell proliferation potential and clone formation 

rate  

 

Figures 3 a and b show the cell proliferation and 

clone formation rate, respectively, of the five groups 

of cells. The cell proliferation and clone formation 



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rate of cells were lowest in A1, but they were 

significantly up-regulated in A2, relative to A3, A4, 

and A5. The differences in cell proliferation and 

clone formation rate among A3 - A5 were not 

significant. Figure 3 c shows the results of cell clone 

formation experiment which indicate that the number 

of clones formed in A1 group was the lowest, while 

A2 had the highest number of clones.  

 

Cell invasiveness  
 

A comparison of the invasion distance of cells in the 

five groups is presented in Figure 4 a. The cell 

invasion distance was significantly shorter in A1 

group than in A2 - A5 groups, while invasion 

distance was longest in A2 group (p < 0.05). Figure 4 

b shows the results of cell invasion experiment in 

each group. The number of cells that invaded in A2 

group was the largest, while A1 had the least number 

of cells that invaded. 

 
 

Figure 4: Cell invasiveness in each group. (a) Cell 

invasion distance; (b) the result of cell invasion 

experiments. *P < 0.05, vs. A1 group; #p < 0.05, vs. 

A2 group  

 

mRNA expression levels of MMP2 and MMP9  
 

Figures 5 a and b display comparison of mRNA 

expression levels of MMP2 and MMP9 amongst the 

five groups. The expression levels of MMP2 and 

MMP9 were significantly lower in A1 than in A2, 

A3, A4, and A5 groups, while MMP2 and MMP9 

levels were significantly higher in A2 than in A3, A4, 

and A5. However, MMP2 and MMP9 levels were 

comparable in A3, A4, and A5 groups. Figures 5 c 

and d show MMP2 and MMP9 band maps, 

respectively. The MMP2 and MMP9 bands of cells in 

the A1 group were the darkest, while those of A2 

group cells were the brightest. 

 
 

Figure 5: Serum bone gla protein levels of rats. (a, b, 

c, and d, are the expression level of MMP2 mRNA, 

expression level of MMP9 mRNA, band map of 

MMP2, and band map of MMP9, respectively). *P < 

0.05, vs. A1; #p < 0.05, vs. A2  

 

Levels of inflammatory factors  
 

As presented in Figure 6, A1 cells had the lowest 

expressions of IL-6, IL-18, and IL-1β, while IL-10 

expression was significantly highest in A1 (p < 0.05). 

The expression levels of IL-6, IL-18, and IL-1β were 

up-regulated in A2, relative to A3, A4, and A5 

groups, but the level of IL-10 was significantly lower 

(p < 0.05). The levels of the four cell inflammatory 

factors were comparable in A3 - A5 groups. 

 

 



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Figure 6: Expressions of inflammation-associated 

factors in the 5 groups. (a, b, c, and d indicate IL-6, 

IL-10, IL-18, and IL-1β, respectively). *P < 0.05, vs. 

A1; #p < 0.05, vs. A2 

 

The expression levels of caspase-3 mRNA and 

protein  
 

There were higher levels of caspase-3 mRNA and 

protein in A1 than in A2, A3, A4, and A5, but 

caspase-3 mRNA and protein were significantly 

lower in A2 than in A3 -A5 groups. However, A3, 

A4, and A5 groups had comparable caspase-3 mRNA 

and protein levels. 

 
 

Figure 7: Expression levels of inflammatory factors 

in the 5 groups. Expression levels of caspase-3 

mRNA (a) and caspase-3 protein (b). *P < 0.05, vs 

A1 group; #p < 0.05, vs A2 group  

 

DISCUSSION  
 

Malignant melanoma is a highly cancerous disease 

which has no specific treatment method. Surgery is 

only effective for treating early-stage melanoma. If 

the disease develops to the middle and advanced 

stages, the prognosis and five-year survival rate of 

patients become greatly reduced [22]. The miRNAs 

are a class of endogenous non-coding single-stranded 

RNAs involved in many cellular processes such as 

organ formation, information transmission, and cell 

growth and division. Studies have revealed that at 

least half of identified miRNAs are associated with 

the occurrence and development of tumors [23]. In 

this study, mouse-derived melanoma cells B16 cells 

were successfully transfected with either miR-22 

mimic overexpression, miR-22 inhibitor, miR-22 

mimic + siNLRP3, or miR-22 mimic NC sequence, 

with un-transfected cells serving as control. These 

data suggest that the transfection with miR-22 cells 

was successful, making the expression level of miR-

22 in cells up-regulated or down-regulated. Analysis 

showed significantly lower NLRP3 mRNA and 

protein levels in A1 group, but the expressions were 

significantly up-regulated in A2 group, relative to 

A3, A4, and A5 groups. The target gene of miR-22 is 

NLRP3. Transfection with miR-22 mimic resulted in 

significant decreases in NLRP3 protein and mRNA, 

while transfection with miR-22 inhibitor resulted in 

significant increases in mRNA and protein 

expression levels of NLRP3. Thus, miR-22, by 

targeting and inhibiting NLRP3, inhibited the growth 

of malignant melanoma cells [24].  

 

Cell proliferation potential and clone formation rate 

of cells were least in A1, but they were significantly 

higher in A2 than in A3, A4 and A5 groups. These 

results indicate that up-regulation of miR-22 

expression level effectively inhibited proliferative 

activity of melanoma B16 cells. This is similar to the 

finding of Houshmandfar et al [25]. In cell clone 

formation experiment, the number of cell clones in 

A1 group was significantly low, while that in A2 

group was higher, in line with the above data. The 

cell invasion assay showed the shortest cell invasion 

distance in A1 group, while cell invasion distance 

was longest in A2 group. This indicated that up-

regulation of miR-22 expression level effectively 

inhibited the invasiveness of melanoma B16 cells. 

The mRNA expression levels of MMP2 and MMP9 

in A1 group were significantly lower than those in 

other four groups, but they were significantly up-

regulated in A2 group, relative to other three groups. 

Both MMP2 and MMP9 are matrix 

metalloproteinases, and they are involved in 

embryonic development, tissue regeneration and 

organ reconstruction [26]. These results indicate that 

up-regulating miR-22 expression level inhibited 

matrix metalloproteinases in melanoma B16 cells and 

regulated cellular events.  

 

There were significantly lower levels of IL-6, IL-18, 

and IL-1β in A1 group than in other groups, while 

that of IL-10 was significantly higher. Thus, up-

regulation of miR-22 expression targeting NLRP3 

inhibition regulated levels of associated inflammatory 

factors and slowed down cell inflammation-induced 

damage. Caspase-3 is a protease and one of the most 

critical enzymes in the apoptosis pathway. It is 

closely associated with the occurrence of cancer, 

aging, and cardiovascular diseases. In this study, 

caspase-3 mRNA and protein levels in A1 group 

were significantly higher than those in other groups, 



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but they were significantly down-regulated in A2 

cells, relative to the other three groups. This indicates 

that up-regulation of miR-22 increased caspase-3 

level, thereby regulating cell apoptosis in melanoma. 

 

CONCLUSION  
 

This study has demonstrated that targeting NLRP3 

inhibition by up-regulating the expression level of 

miR-22 significantly reduces the multiplication, 

invasiveness, and matrix metalloproteinase activities 

of melanoma B16 cells, with adverse impact on the 

occurrence and development of malignant melanoma. 

However, although the expression levels of 

inflammatory factors and mRNA and protein levels 

of caspase-3 were assayed, their correlation with up-

regulation of miR-22 expression levels targeting 

NLRP3 inhibition was not further investigated. The 

experiments in this study were performed only at the 

cellular level. No in vivo (animal experiments) or 

clinical experiments were carried out for more in-

depth investigations. However, the results provide 

some useful reference data for the treatment of 

malignant melanoma at the gene level. 

 

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