The Role And Mechanism Of MiR-145-5p In Occurrence And Development Of Malignant Melanoma

Malignant melanoma(MM)is a common tumor type,and mainly occurs in the skin tissue,secondly in the mucous membrane and eye.Malignant melanoma with its propensity to metastasize and poor prognosis has higher incidence among people with light-colored skin.Malignant melanoma is a disease caused by many factors,including environmental and genetic factors.Therefore,it is extremely important to study the role of gene expression and regulation in the development of melanoma.MicroRNAs(miRNAs)are a kind of non-coding RNAs,which can lead in the degradation of specific mRNAs,or inhibiting the translation of proteins by binding to the 3’-UTR region of the target mRNAs.Several studies have found that miRNAs can affect the occurrence and development of melanoma.miR-145-5p has been reported as a tumor suppressor gene in numerous cancers,such as gastric cancer,laryngeal squamous cell carcinoma,and prostate cancer.It has been found that miR-145-5p inhibits epithelial mesenchymal transformation through targeting N-cadherin and ZEB2,thereby inhibiting the invasion of gastric carcinoma.Moreover,the expression of miR-145-5p is closely related to Lauren’s classification,tumor infiltration depth,lymphnode metastasis and TNM stages in gastric cancer.It has been reported that miR-145-5p levels are significantly decreased in laryngeal squamous carcinoma,and low expression of miR-145-5p can repress the target gene FSCN1 expression and promote cell proliferation,invasion,metastasis,and interstitial transformation,which functions as a tumor suppressor.In prostate cancer,the miR-145-5p overexpression can supress expression of SOX2,thus inhibiting the proliferation of prostate cancer cells.Some studies have found that expression of miR-145-5p is significantly reduced in melanoma tissues compared with paracancerous tissues,and miR-145-5p can affecting proliferation and apoptotic migration and invasion of melanoma cells by inhibiting the MAPK and PI3K/Akt pathways through targeting NRAS.It has been proved that TLR4 expression in malignant melanoma is significantly up-regulated,compared with normal skin tissue,and activation of TLR4 by lipopolysaccharide(LPS)obviously promotes Foxp3 expression,which is involved in the immune escape in malignant melanoma.According to prophase studies in this laboratory,TLR4 was predicted to be a target gene of miR-145-5p through bioinformatics softwares Targetscan and picTar,miRanda.So far,the role and mechanism of miR-145-5p in the occurrence and development of malignant melanoma by targeting TLR4 hasn’t been studied.According to several studies,the NF-κB signaling pathway is related to biological processes of a variety of cancers.For example,in gastric cancer,siRNA has been found to inhibit gastric cancer SGC-7901 cells proliferation and invasion through blocking the NF-κB signaling pathway.In hepatocellular carcinoma,Embelin inhibits proliferation and promote apoptosis in Bel-7404 cells by blocking the NF-κB signaling pathway,thereby inhibiting the development of the liver cancer.At present,the research about whether miR-145-5p could affect the occurrence and development of malignant melanoma by regulating the NF-κB signaling pathway hasn’t been reported so far.In order to study the roles and mechanisms of miR-145-5p in occurrence and development of malignant melanoma,firstly the miR-145-5p expression in malignant melanoma was detected by qRT-PCR,and the relationship between miR-145-5p expression and clinicopathologic parameters was analyzed;this study explored the effects of miR-145-5p on the melanoma cell proliferation,colony formation,apoptosis,migration and invasion,the relationship of miR-145-5p and TLR4 was verified by using Dual luciferase reporter gene assay,and the effects of miR-145-5p on the cell biological behavior and the NF-κB signaling pathway by targeting TLR4 were investigated;finally in vivo,A375 melanoma xenograft model in nude mice were established to explore the effect of miR-145-5p overexpression on the TLR4 expression,xenograft growth and the NF-κB signaling pathway.This study is divided into the following three parts:Part Ⅰ: Study on the expression of miR-145-5p and its significance in malignant melanoma;Part Ⅱ: Study on the regulatory effect of miR-145-5p on the biological behavior of malignant melanoma cells;Part Ⅲ: Study on the anti-tumor effect of miR-145-5p on melanoma cells xenograft in nude mice.Main content:Part Ⅰ: Study on the Expression of miR-145-5p and its Significance in Malignant melanoma Methods1.The expression difference of miR-145-5p in malignant melanoma and normal tissues was detected by qRT-PCR.2.The expression of miR-145-5p in melanoma at different stages was detected by qRT-PCR.3.The relationship between miR-145-5p expression and clinicopathological parameters was analyzed in malignant melanoma.Results1.miR-145-5p expression was significantly in malignant melanoma compared with normal tissues.2.The expression level of miR-145-5p in melanoma at stage1/2 was significantly higher than that in melanoma at stage3/4.3.The positive expression rate of miR-145-5p in melanoma was not significantly related to the patient’s gender,age,tumor location,and ulcer,and was related to the melanoma stage.Part Ⅱ: Study on the Regulatory Effect of miR-145-5p on the Biological behavior of Malignant melanoma cells Methods1.The lentivirus vectors containing miR-145-5p,sh-miR-145-5p,or TLR4 were constructed,viral packaging was performed and then lentiviruses were titrated.2.The expression of miR-145-5p in Melanocytes and melanoma cell lines(A375,WM35,VMM5 A,M14,A875 and HMCB)was detected by qRT-PCR.3.The recombinant lentiviruses infected A375 and M14 cells,and infected cells were screened to gain stable cell lines.4.The proliferation,clonal formation,migration,apoptosis and invasion of A375 and M14 cells transfected with miR-145-5p,sh-miR-145-5p or miR-145-5p + TLR4 were detected using MTT,clonal formation,scratch,flow cytometry,and Transwell chamber assays.5.The activity of Caspase3 and NF-κB in A375 and M14 cells transfected with miR-145-5p,sh-miR-145-5 or miR-145-5p+TLR4 was determined by ELISA assay.6.Online bioinformatics softwares predicted the target relationship between miR-145-5p and TLR4.The target relationship between miR-145-5p and TLR4 was confirmed by Dual luciferase reporter gene assay.7.The effect of miR-145-5p on expression of TLR4 mRNA in A375 and M14 cells was detected by qRT-PCR.8.Western blot was used to detect expression of the TLR4,NF-κB p65,MMP-2,MMP-7 and Ki67 proteins in A375 and M14 cells transfected with miR-145-5p,sh-miR-145-5 or miR-145-5p+TLR4.Results1.qRT-PCR results showed that compared with normal melanoma cells,the miR-145-5p expression in melanoma cell lines(A375,WM35,VMM5 A,M14,A875 and HMCB)was significantly decreased,especially in A375,M14 cells.Therefore,A375 and M14 cell lines were choosed as cellular materials in this study.2.Expression of miR-145-5p was significantly increased in the A375 and M14 cells transfected with miR-145-5p compared with control group,whereas a significant reduction in expression of miR-145-5p was determined in A375 and M14 cells transfected with sh-miR-145-5p,indicating that overexpressed and interfered miR-145-5p cell lines were successfully constructed.3.In A375 and M14 cells,miR-145-5p overexpression significantly inhibited cell proliferation,clone formation,invasion,migration,the NF-κB activity,the expression of the NF-κB p65,and promoted cell apoptosis.miR-145-5p downregulation obviously promoted cell proliferation and clone formation,invasion,migration,the NF-κB activity,the expression of the NF-κB p65,and inhibited apoptosis.4.The online softwares Targetscan and picTar,miRanda predicted that TLR4 was the target gene of miR-145-5p,and the Dual luciferase reporter gene assay confirmed that miR-145-5p directly binded to TLR4.miR-145-5p overexpression significantly inhibited the expression of TLR4 in A375 and M14 cells,while miR-145-5p interference could promote the expression of TLR4.5.In A375 and M14 cells,miR-145-5p overexpression inhibit the expression of TLR4,cell proliferation,clonal formation,invasion and migration,inhibit the activity of NF-κB and the expression of NF-κB p65,MMP-2,MMP-7 and Ki67 proteins,and promote cell apoptosis by targeting TLR4.Part Ⅲ: Study on the Anti-tumor Effect of miR-145-5p on Melanoma cells Xenograft in Nude mice Methods1.The xenograft models were constructed by transplantation of melanoma cell A375,and the volume of xenografts was measured regularly,and weight of xenografts was detected 30 days later.2.Western blot was used to measure expression of TLR4 and NF-κB p65 proteins in xenograft.3.The effect of miR-145-5p overexpression on the NF-κB activity in xenograft was detected by ELISA.Results1.The Con,miR-145-5p melanoma xenograft models with A375+miR-con,A375+miR-145-5p were constructed successfully.The results from tumor volume and weight detection indicated that miR-145-5p overexpression significantly inhibited the growth of melanoma xenograft.2.The miR-145-5p overexpression inhibited the expression of TLR4 and the activity of the NF-κB signaling pathway.ConclusionCompared with normal tissues,miR-145-5p expression was significantly decreased in malignant melanoma tissues.The expression level of miR-145-5p in melanoma was related to the melanoma stage.In A375 and M14 cells,miR-145-5p inhibited cell proliferation and clone formation,migration and invasion,and promoted apoptosis through inhibiting the activity of the NF-κB signaling pathway by targeting TLR4.The further study in vivo showed that miR-145-5p overexpression inhibited the activity of the NF-κB signaling pathway by targeting TLR4,thereby inhibiting the growth of malignant melanoma,which provided a certain experimental basis and theoretical basis for the clinical treatment of melanoma.