Alternative Splicing Of Smad4 And Its Function In HaCaT Cells In Response To UVB Irradiation

The skin is the outermost,largest organ of the body and is the largest contact area with the external environment such as sunlight and pollution.The skin protects the body from dehydration,pathogens and external mutagens(heat,sunlight,injury).The skin has the function of regulating body temperature,storing fat,water,and vitamin D.The skin consists of epidermis,dermis and subcutaneous tissue.Excessive exposure to ultraviolet(UV)radiation is known to be one of the risk factors for carcinogenesis of skin cancer,and pathogenesis is a hot topic in biology and clinical research.Recently UV-induced gene-selective splicing has received increasing attention from researchers.In the United States,skin cancer is one of the three most common types of cancer.It is estimated that there are 56 cases of skin cancer patients per 100,000 men and 61 cases of skin cancer patients per 100,000 women.The proportion of men and women is 1.5-2: 1.Skin cancer is called skin malignancy,according to the different origins of tumor cells have different classification and naming,such as squamous cell carcinoma,basal cell carcinoma,malignant melanoma and so on.Skin squamous cell carcinoma,formed by flattened squamous cells at the top of the epidermis.Basal cell carcinoma,which is formed in rounded basal cells located under squamous cells.Malignant melanoma is formed in melanocytes that are present in the epidermis.Among them,skin squamous cell carcinoma and basal cell carcinoma is also known as non-melanoma skin cancer,is the most common type of skin cancer.The incidence of squamous cell carcinoma and basal cell carcinoma in non-melanoma skin cancer was 4: 1.Most basal and squamous cell carcinoma can be treated.the condition can be controlled.It is easier to cure melanoma.If a tumor investigates before spreading to the dermis(inner layer of the skin).Melanoma early detection and treatment is unlikely to lead to death.Despite the increasing number of sunscreens and awareness,70-90% of basal cell carcinoma(BCC)developed in sun-exposed head and neck regions,with 5% to 10% of all skin cancers occurring in individual eyelids.Particularly,33610 cases of eyelid BCCs were recorded between 2000 and 2010 at England.Ultraviolet radiation is considered to be one of the most likely causes of skin cancer,although people’s awareness of protection gradually increased,and take some effective sun protection measures,but the incidence of skin cancer is increasing year by year.In the area of the eyelid,the medial cornea at the midpoint of the upper eyelid and lower eyelid has been shown to be not only a particularly common site of BCC but also about poor prognosis.It is speculated that the high incidence of non-melanoma skin cancer on the eyelids is due to the thinnest on the body of the skin,which is particularly susceptible to prolonged UV light damage,which is a defined risk factor BCC and Squamous Cell Carcinoma.This group of cancer contains basal cell carcinoma and cutaneous squamous cell carcinoma.Fortunately,the mortality linked with nonmelanoma skin cancer is unusual.However,malignant melanoma accounts for 75% of the skin cancer-related deaths.Exposure to ultraviolet radiation(UV)radiation is one of the risk factors for carcinogenesis of skin cancer,but it is unclear for the pathogenesis of UV-induced skin cancer,which is also a hot topic in biology and clinical research.In nature,the main source ultraviolet light is the sun,produced electromagnetic radiation having wavelength 10-400 nm.According to the wavelength,UV can be divided into the long-wavelength ultraviolet(UVA 320-400 nm),(UVB 290-320nm)and short wavelength(UVC 100-290nm).The ozone layer is vital to protect the world from dangerous radiation.However,nowadays the ozone layer is disturbed and damage.The oxygen and ozone layer are essential to absorb UVC radiation entirely and approximately 90 percent of UVB.Due to the shorter wavelength UVC,is entirely absorbed by the ozone layer of the atmosphere and not reach the earth surface.Therefore,the most harmful UV rays are UVA and UVB.UV radiation is an important cause of the induction of skin cancer.The excessive exposure to UVB and UVA cause various type of DNA damage.The DNA damage may have numerous harmful consequences like mutation,cell death,photoaging,and cancer.UVA and UVB are not only absorbed by DNA but also by the chromophore which is present in the skin.This process may result in DNA damage indirectly due to the formation of reactive oxygen species.However,the human cells have a complex system to control the harmful effects of reactive species by antioxidant and DNA damage repair mechanism.UVB is the main source of Vitamin D which maintain calcium hemostasis and another important process.The DNA damage will activate the cell cycle checkpoint,cell cycle arrest phenomenon,and thus induce apoptosis mechanism.In addition to induced DNA damage,UV radiation also causes the activation of the proto-oncogene,inactivation of tumor suppressor gene,causing immune suppression,activation of intracellular signal transduction pathway.In recent years,ultraviolet radiation caused variable gene splicing is the focus of researchers.The study found that the formation of UV-induced gene mutant splice,the occurrence,and development of the tumor has a certain regulatory role.Excessive exposure of skin to UVB cause skin cancer,including basal cell carcinoma,squamous cell carcinoma,nonmelanoma and cutaneous malignant melanoma.Literature study reveals that UVA and UVB wavelength triggers DNA damage and responsible of the majorities of lesion and cancers.The two major DNA lesion appear due to UV Irradiation,cyclobutane pyrimidine dimmer(CPDs)and 6-4 pyrimidine photoproducts(6-4 pps).The removal of CPDs is carried out by a complex and highly conserved mechanism called nucleotide excision repair(NER)in placental mammals.The loss of function of mutations in the NER regulatory pathway has been shown to result in a dramatic increase in cutaneous carcinogenesis in patients with Xeroderma pigmentosa patients.Keratinocytes have a defensive mechanism known as a DNA damage(DDR)response against cutaneous carcinogenesis.Which may have different physiological results such as cell cycle arrest,activation of DNA,apoptosis or senescence: The activation of this signaling pathway may have an important relationship with the formation of skin cancer,in this part,we will explore the UV can be activated signaling pathways,focusing on UV irradiation and TGF-β/Smad signaling pathway.TGF-β singling pathways start by binding TGF-β/BMP ligands to a pair of membrane receptors(TBRII+TBRI/ BMPRII+BMPRI).These receptors phosphorylate R-Smads(2,3 and 1,5,8)to promote the establishment of the heterotetrameric receptor complex.The R-Smads interact with Co-Smads(Smad4),which acts as a shuttle between nucleus and cytoplasm.The Samd4 and associated R-Smads translocate to the nucleus and bind to transcription factor to regulate gene expression.Their function with many fine physiological processes related to cellular and tissue,including immune responses,inflammatory responses,developmental processes,cellular growth,proliferation,hemostasis,and apoptosisThe transforming growth factor beta(TGF-β)is a member of a large family of structurally related proteins that normally function to regulate embryonic development and cell homeostasis,including the cell proliferation regulation,differentiation,matrix production and apoptosis in a cell.Alteration in the transforming growth factor beta(TGF-β)signaling pathway can cause human diseases,for example,developmental disorders,vascular disease,and cancer.The TGF-β acts as a tumor-suppressor in the early stages of epithelial carcinogenesis,but TGF-β induces tumor development in advanced phases by inducing tumor growth,epithelial-mesenchymal transition(EMT),invasion,and metastasis.As a tumor suppressor gene,the function and variants of Smad4 have been well studied.SMAD4 is a member of the SMAD family and plays a key role in mediating members of transforming growth factors β superfamily signal transduction and gene regulatory events.It is an important tumor suppressor.The possibility of the loss of BMP,TGF-β signaling components and TGF-β-type II receptors is contributing to the development or progression of advanced meningiomas.In the early stage of normal epithelial cells and tumorigenesis,TGF-β is antiproliferative,but plays a role in tumor promoters at a later stage,assisting in metastatic progression through an autocrine TGF-β loop.The growth inhibition properties of TGF-β are paramount and demonstrated in normal and early tumor cells.This property is due to the suppression of c-Myc proto-oncogene or cyclin-dependent kinases(CDK)and even inhibition of CDK regulators such as p15,P21,and p27.The TGF-β and other proteins that involved in TGF-β signaling pathways initially express in most of the cancers.However,during transfection of TGF-β into tumor cells,the tumor cells show resistance to the inhibitory effect of TGF-β and leading to cell proliferation,invasion and increases metastasis.The inactivation of some components of TGF-β signaling pathways such as TBRI,TBRII,and Smads contribute to metastasis,cell proliferation,and cancer development.The TGF-β overexpression increases angiogenesis,metastasis,invasiveness and immune suppression.The overexpression of TGF-β has been detected in various tumors such as colon,breast,esophageal,gastric,liver,lungs,pancreas,kidney,brain,prostate,malignant melanoma and certain blood disease.TGF-β increase epithelial cell motility and prompt metastasis development.The study of TGF-β signaling pathways and Smads effects is critical for certain diseases such as cancer.Alternative splicing(AS)is one of the most common mechanisms of human gene regulation and plays a crucial role in increasing the diversity of functional proteins.Many diseases are linked alternative splicing,especially cancer.Recently,more and more studies have focused on the relationship between AS and cancers.The splicing variants of Smad4 have been found in many cancers.Alternative splicing occurs in almost all human genes.Approximately 95 percent of the human multi-exon genes are spliced alternatively.Cancer is a complex disorder which is associated with a variety of genetic and epigenetic aberrations.Cells acquire eight common characteristics during cancer development.They include maintenance of proliferative signaling,elimination of growth suppressor,resistance to cell death,the possibility of replicative immortality,induced angiogenesis,and activation of invasion and metastasize.Many of these functions may be related directly resulting in abnormal gene regulation,dysregulation of alternative splicing during tumor progression.The destruction of TGF-β signaling network at Smad level is commonly found in human cancers.Several sporadic epithelial tumors show missense,nonsense,frameshift,and point mutations at SMAD4 locus.The four alternatively spliced tumors associated Smad4 isoform have been found in thyroid tumor.Smad4 is often mutated and deregulated by an abnormal splicing in thyroid cancer.The present work determined the alternative splicing of Smad4 in Ha Ca T cell in response to UVB irradiation.Different types of alternative splicing exist,in which introns are detached,and exons are rejoined together,including mutually exclusive splicing,splicing alternative 3 ’or 5’ splice sites,and exon skipping,the most common is intron retention.Alternative splicing allows for significant amounts of protein diversity in humans with different functions in cellular processes,developmental processes,and disease states.The number of proteins encoding genes in mammals is about 20,000 to 35,000,but the proteins number is much higher due to alternative splicing.Alternative splicing limits binding characteristics,protein stability,enzymatic activity,post-translational modifications and intracellular localization of various proteins.Many proteins have different domains with variable function can be used protein-protein interaction or enzymatic activity.The exons correspond alternative splicing can mix the protein domains which affects the nature and function of proteins.The two main categories of alternative splicing are protein level and transcript level.Alternative splicing which produces abbreviated protein due to a frameshift and exclusion or inclusion of specific exons having different function such type of splicing is called protein level.In the transcript level,it produces different splice having different translation ability.Thus,it takes part a significant role in expanding protein diversity.The current RNA sequencing data showed that more than 95% of human genes yield at least two pre-m RNA and an average of seven m RNA isoforms from a single gene.There are several methods for the detection of splice variants,but computational prediction and microarray analysis is a most effective method.The microarray is the most popular,robust and accessible method for detection of splicing variants.RT-PCR and nested PCR can also be used for identification of gene isoform.Several computational methods and software have been developed to detect exon-skipping events,such as ASprofile Diff Splice,and DSGseq.The computational prediction method based on genome alignment and transcript sequences.The popular programs to perform pairwise alignment of c DNA and genomic fragments include SIM4,Spidey,EST_GENOME,MGAlign,and Gene Seqer.The expressed sequence Tag(EST)was the first computational approach to detect AS events.The computational prediction approach can only use for analysis of already available splice sequence,not for novel isoform.Microarray-based methods were used to detect a number of the novel gene transcript.Which was not detected by EST using the computational method.There are two DNA microarray,glass c DNA microarray,and oligonucleotide microarray.The glass c DNA microarray is relatively cheap and increased detection sensitivity while oligonucleotide microarray is specific,fast,reproducible and very attractive for large scale of studies.The Glass CDNA have some disadvantages like the requirement of intensive labor for purifying,synthesizing and storing of DNA solution.While the oligonucleotide array format is still expensive required some specialized equipment for washing of unbound sample,hybridization,staining of label and quantification process.The splicing detection of a gene on microarray-based have some unique challenges in probe designing for isoform detection having a high grade of homology.A combined probe for exon and exon-exon junction can be used for microarray-based splice detection.The following two approaches in probe designing to detect RNA splicing by DNA microarray;exon and tiling arrays.The tiling arrays have more ability to detect new splicing and known events.Allele ID? can be used for designing of the junction as well as intra-exon probes.RT-PCR and Nested PCR is a better method to detect splicing is increasing multiplexed,highly precise and sensitive but not genome-wide.Alternative splicing events are linked to numerous human diseases like cardiovascular diseases including cancer,respiratory diseases,neurodegenerative diseases,and metabolic diseases.Multiple activities of tissues like the brain require many different proteins with dispersal function.The m RNA splicing can alter the protein composition leading to many neurological processes.Point mutations cause several human diseases such as ataxia-telangiectasia and neurofibromatosis.Half of the patients are suffering from the disease as a result of a mutation in pre-m RNA.In some splicing disorders,the regulatory factor which is essential for splicing process become mutated and disturb splicing activity.The mutation of splicing factor altered pre-m RNA splicing.It can lead to severe disorders or mild disease.Alternative splicing generates a wide variety of protein isoforms with unique functions having different ability to react with a variety of pharmaceutical products.Alternative splicing participates in the regulation of normal physiological functions and the disease.The percentage of abnormal spliced m RNA is high in cancer cells.This thesis aims to describe the general aspect of Smads family,alternative splicing process,the universal aspect of the Smad4 alternative splicing of cancer and the possible use of spliceoform for the diagnosis and therapeutic purpose.The main purpose and objective of this research are to highlight specific mechanisms involving alternative splicing of cancer and to show new evidence of alternative splicing in different steps giving the initiation and progression of cancer.The contents include:(1)The effect of UVB on Smad4 alternative splicing and its function in Ha Ca T cell line.(2)The effect of Smad4-FL overexpression and Smad4 B on cell survival pre-and post UVB irradiation.(3)The involvement of splicing factor(hn RNPA1 and SF2)in UVB induced alternative splicing of Smad4.(4)The effect of different doses and time on Smad4 expression and alternative splicing.The present study determined alternative splicing of Smad4 and its function in Ha Ca T cell lines in response to UVB irradiation.Ha Ca T cells were treated with different doses of UVB after 6 hours incubation.Nested PCR was performed to detect Smad4 isoform in UVB irradiated Ha Ca T cell.The given result reported one novel isoform Smad4 B Ha Ca T cell in response to UVB Irradiation at 6 hours.The UVB induced Smad4 B isoform led to decreased the Smad F4 expression.The Smad4 B support Smad4 F in UVB resistance within certain limits.At high dose of UVB irradiation Smad4 F completely knockdown while Smad4 B expression increased.The result showed that Smad4 B act as the best supporter of Smad4 against UVB.The same isoform was also reported at a low dose of UVB(3,5,7 mj/cm2)at 24 hours incubation.There was no significant difference between Smad4 F and Smad4 B on cell proliferation.The given isoform decreases the expression of N-cadherin while smad4 overexpression increases the expression of N-cadherin.The given isoform was first time reported.The splicing factors are essential proteins that regulate gene alternative splicing,which leads to the production of genetic variants,which in turn affects the physiological processes of cells.The heterogeneous nuclear ribonucleoprotein(hn RNP)family proteins and prem RNA splicing factor ASF/SF2/SRSF1 regulate pre-m RNA splicing effectively.Previously it has been reported that hn RNPA1 and SF2 regulate gene alternative splicing.The overexpression of hn RNPA1 and ASF/SF2/SRSF1 has been recognized in many cancer cells denote its relation with the motility and proliferation of cancer cells.The present study determines whether the splicing factor(hn RNAP A1 and SF2)is responsible for regulating UVB induce alternative splicing of Smad4.The heterologous nuclear ribonucleic acid A1/A2(hn RNP A1/A2)and the splicing factor 2/alternative splicing factor(SF2 /ASF)are the keys to splicing m RNA(prem RNA)splicing.Many studies have demonstrated that some RNA-binding proteins can participate in inflammatory processes and the regulation of tumorigenesis by regulating the splicing or m RNA stability of inflammation and tumor-associated genes.The two nuclear RNA binding proteins,the heterologous nuclear ribonucleoprotein family(hn RNP)and the serine/arginine-rich protein(SR)family play a key role in regulating alternative splicing and m RNA stability.The hn RNP family comprises at least twenty members,primarily bind to the sequence of splice silencers,which are located in exons(ESSs,exon spliced silencers)or introns(ISSs,intron splice silencers)to promote exclusion of exon and act as splice repressor.The most copious and unique proteins in this group are hn RNP A1 and hn RNP A2,with high sequence homology and functional homology.The growing evidence that hn RNP A1 and hn RNP A2 are overexpressed in various tumors and are used as early tumor biomarkers.The SR protein family and the other regulator for the alternative splicing are also more than 20 members.These proteins bind to splice enhancers in exons(ESEs,exon splicing enhancers)or introns(ISE,intron splice enhancers)and are primarily used as antagonists of hn RNP protein.However,some studies have also shown that SR proteins regulate exon cleaving events,and different SR proteins exhibit opposite action when promoting exon skipping or inclusion of the same gene.It is reported that the splicing factor 2/alternative splicing factor(SF2 / ASF),which is the best individual member of the SR family,is upregulated in a variety of human cancers(including lung and cervical cancer)and plays an important role in the establishment process maintain cell transformation.The presence of the Arg / Ser-enrichment domain is uniqueness that distinguishes the SR protein from other RNA-binding proteins.This signed RS domain also exists in other proteins that carry several other types of protein domains,for example,Zn finger domain or RNA helicase domain,which is commonly referred to as SR-related protein.One important feature of SR proteins is their extensive selfregulation and cross-regulation to control the expression of individual SR proteins in cells.Since some names of the same SR protein has been given in the course of their discovery,a new standardized nomenclature has been proposed for the "core" SR protein family,which consists of 12 relatively well-characterized members Composition.For example,SF2/ASF or ASF/SF2 becomes SRSF1;SC35 is renamed to SRSF2;SRp20,SRp40,SRp55,SRp75,and 9G8 are called SRSF3 to SRSF7,respectively.The splicing community has steadily adopted this new vocabulary,although other structural and functionally related proteins,such as human Tra2α/β and RNPS1,may finally join this core once their splicing activity is further characterized.The given study was focused on two well-known splicing factors,namely hn RNP A1 and ASF /SF2/SRSF1.The effects of hn RNPA1 and ASF/SF2 on the alternative splicing of Smad4 induced by UVB radiation were analyzed by using cellular and molecular biology methods.The result showed that hn RNPA1 is responsible for alternative splicing of Smad4 in response UVB irradiation.The UVB irradiation influence on hn RNPA1 and SF2 expression.The given study reported that UVB increase the expression of hn RNPA1 and SF2.The overexpression of hn RNPA1 is responsible for alternative splicing of Smad4 and led to Smad4 B isoform.The UVB increase the expression of hn RNPA1 splicing factor which causes Smad4 alternative splicing resulting Smad4 B isoform.This isoform is shown more resistance to UVB as compared to Smad4.The resulted Isoform decrease the expression of N-Cadherin.