The Role Of YTHDF1 In Regulating IL-8 In Proliferation,Migration And Drug Resistance Of Environmental Factors-related Squamous Skin Cancer Cells

Objective:The skin is an organ that is the most exposed to the external environment and its integrity and extent of interaction with environmental factors affect the health of organisms.Skin squamous cell carcinoma?SCC?is the second most common skin malignancy.It is an invasive cancer,have a very strong vicious trend.Compared with other types of skin cancer,it proliferates faster,has certain drug resistance,has a high metastatic rate,it is also easy to metastasize to lymph nodes,liver,lungs,and even life-threatening.There are many external factors that can cause skin cancer,such as,ultraviolet radiation,chemical carcinogens,ionizing radiation,viruses and other diverse environmental carcinogenic factors.And it's also closely related to genetic characteristics,immune function,and hormone levels.Epigenetic modification is an important modification in the pathogenesis of skin cancer.RNA methylation mediated epigenetic transcriptome regulation and function has become a new field of RNA biology.The newly emerging RNA epigenetic inherited N6-methylated adenine as a marker to study the regulation of eukaryotic gene expression after transcription.RNA methylation modifications are regulated in vivo by three modified proteins,named writer,eraser and reader.The reader is the m6A binding protein.The currently reported proteins that specifically recognize and bind to m6A are mainly members of the YTH-domain family.It mainly including YTHDF1,YTHDF2,YTHDF3,YTHDC1,YTHDC2,etc.Among them,YTHDF1 will improve translation efficiency and actively promote protein synthesis through interaction with translation machines.Interleukin-8?IL-8?,a cytokine of the chemokine family,also named as chemokine CXCL8,it is an inflammatory chemotactic factor secreted by macrophages and epithelial cells.The main biological activity of IL-8 is to attract and activate neutrophils.The neutrophils will undergo morphological changes after contact with them,and these effects may lead to local inflammatory reactions.Studies have shown that IL-8 shows overexpression in melanoma and its cognate receptors CXCR1 and CXCR2.Moreover,IL-8 can activate AKT by binding to the corresponding receptor to activate AKT,which leads to a series of signal cascades.After phosphorylation of AKT,it activates downstream GSK-3?to regulate cell proliferation,apoptosis and migration.Therefore,the aim of this study was to observe the changes in the content of YTHDF1 in normal skin cells and squamous skin cancer cells,and to clarify the role of YTHDF1 in the growth of tumor cells,and to analyze the proliferation,migration and drug resistance of squamous skin cancer cells after YTHDF1 knockout.The influence of apoptosis and the regulation of IL-8 in squamous skin cancer provide theoretical and experimental basis for further study on the regulation of YTHDF1 on cells,and provide new ways to prevent and treat skin cancer through the idea of methylation.Methods:1.Select human squamous skin cancer cell A431 cells,establishing a cell homeostasis model of YTHDF1 Knockout.Then get A431 sh-GFP and A431 sh-YTHDF1 two groups cells.Incubate two groups cells with 10%fetal bovine serum,1%penicillin-streptomycin in complete medium,5%CO₂ in a constant temperature cell incubator.The cells were divided into A431 sh-GFP and A431 sh-YTHDF1 groups for a series of cell function tests,including cell proliferation,migration,drug resistance,and apoptosis.First,the protein expression of the two cells was detected by Western blot.Then use CCK8 to detect cell proliferation.Two groups of A431 sh-GFP;A431sh-YTHDF1 cells were seeded on a 96-well plate,after 24 hours added CCK8 10?l,and1 hour later,the absorbance was measured at 450 nm on a microplate reader to obtain proliferation data.Prepare the two groups of cells for apoptosis detection according to the instructions of the apoptosis kit,and then perform the detection on the flow cytometer.After 24 hours cell culture,chemotherapeutic drugs pentafluorouracil 5-fu?concentrations of 1?M,2?M,4?M,8?M?and DDP?concentrations of 5?M,10?M,20?M,40?M?.The same method was used to measure the absorbance at 450nm of the microplate reader,and the data of the two groups of cells were analyzed by analyzing the data.Then start wound healing experiment.Cells were seeded into six well plates at a density of 5×10⁵/well.The next day,the tip of gun is scratched perpendicular to horizontal line on the back.Take photos at a certain time,calculate scratch rate and cell migration rate from field of view photos.Detect the content of IL-8 in the supernatant of two groups of cells by ELISA experiment;Detection of IL-8 m RNA expression in two groups of cells by total RNA extraction and Real-time PCR.At the same time,changes in cell stability were also detected at three time points:0h,3h,and 6h after adding actinomycin D;Added IL-8recombinant protein to A431-GFP and A431 sh-YTHDF1 cells at a concentration of 1?g/ml.Get four groups of cells?A431 sh-GFP,A431 sh-YTHDF1,A431 sh-GFP+IL-8,A431 sh-YTHDF1+IL-8?.The previous series of cell function tests were repeated for the four groups of cells,and the role of IL-8 factor in the development of squamous skin cancer was analyzed based on the results of recovery experiments.Result:1.The results show that HaCaT cells treated with UVB and arsenic can increase the expression level of YTHDF1 protein.Two normal skin cells?HaCaT cells and NHEK cells?and two cells?A431 cells and PAM212 cells?with squamous skin cancer were compared and found to have a significant increase in YTHDF1 content in squamous skin cancer cells.2.Compared with control group A431 sh-GFP,the results of 24h proliferation experiment showed that the proliferative ability was weakened after knocking out YTHDF1?P<0.01?.The results showed that the apoptotic rate of A431 sh-YTHD1group was higher than that of non-knockout A431 sh-GFP group?P<0.01?.The scratch test showed that the migration ability and scratch healing ability of the cells were weakened after knocking out YTHDF1.In the drug resistance experiment,CCK8 and flow pattern were withered.The two methods of death showed that the number of cells in both groups gradually decreased with the increase of drug concentration and the drug resistance of A431 sh-GFP group was higher than that of A431 sh-YTHDF1?*P<0.05?.3.After sequencing the downstream influence factors of A431 cells,get the sequencing map and the volcanic map.Among the many influencing factors,IL-8 factor played a greater role in the occurrence of squamous skin cancer than other factors.The expression of IL-8 inflammatory factors in the supernatant of two A431 cells was detected by ELISA kit.The expression of IL-8 in the un-knocked YTHDF1 group was higher than that in the knockout group?P<0.01?.RT-The expression level of IL-8 in the A431 sh-GFP group was consistently higher than that in the A431 sh-YTHDF1 group?P<0.05?.The stability of actinomycin D also showed that the expression level of IL-8m RNA in the A431 sh-GFP group was always higher than that in the A431 sh-YTHDF1group.And with the increase of actinomycin D treatment time,the stability of IL-8decreased between the two groups.4.Detection of four different cell functions in cell recovery experiments:proliferation,migration,drug resistance,and apoptosis.In four groups of cells?A431 sh-GFP,A431sh-YTHDF1,A431 sh-GFP+IL-8,A431 sh-YTHDF1+IL-8?,When YTHDF1 and IL-8 coexist?A431 sh-GFP+IL-8 group?,proliferative ability is stronger than the other three groups,migration speed and scratch repair rate are higher than the other three groups,the degree of drug resistance and apoptosis are also the most expressive of the four groups.Instead,When neither YTHDF1 nor IL-8 is present?A431 sh-YTHDF1group?,the expression ability of the above four functions is shown to be the weakest in the four groups.And the difference with the other three groups has a certain statistical significance.At the same time,it was found that after the IL-8 recombinant protein was added,compared with the previous two groups of A431 sh-GFP and A431 sh-YTHDF1cells,their corresponding migration,proliferation,resistance increased,and apoptosis decreased,and all have a certain statistical significance.Conclusion:1.UVB and arsenic exposure increase the content of YTHDF1 in skin cells.2.YTHDF1 increases the proliferation,migration and drug resistance of squamous skin cancer tumor cells.3.YTHDF1 can regulate IL-8 to play a role in the growth of squamous skin cancer cells.