Ⅰ:MiR-210-3p And MiR-630 Inhibit Lung Cancer Cell Proliferation And Its Mechanisms.Ⅱ:Identification Of A Novel Transcript Isoform Of The TTLL12 Gene In Human Lung Cancer

Part I: Mi R-210-3p and mi R-630 inhibit lung cancer cell proliferation and its mechanismsBackground Lung cancer is the most common cancer worldwide with poor 5-year survival rate nowadays.Though many important tumor suppressor genes and oncogenes for lung cancer have been identified,there are still a number of lung cancer patients diagnosed at later stages and died without successful therapies.Thus,more studies on critical genes and their function network for lung cancer development are pivotal for early diagnosis and detection of novel therapeutic approaches for lung cancer.micro RNAs,a group of small,single-stranded and endogenous non-coding RNAs,have emerged as crucial factors that post-transcriptionally regulate gene expression in many human diseases including cancers.There are accumulated evidences that mi RNAs do very important roles in the regulation of tumorgenesis,tumor growth,tumor migration and invasion.By recognizing the short fragment of m RNA with imperfect complementarity and silencing the expression of target genes,mi RNAs can degrade or suppress the target m RNA transcription.mi R-210 is well known to be a sensitive and specific tumor biomarker for lung cancer diagnosis.By measuring the expression of mi R-210 in lung tumor tissues and sputum,the early-stage non-small lung cancer(NSCLC)could be detected sensitively.Some studies suggested that mi R-210 acts as an onco-mi RNA in NSCLC patients,as the up-expression of mi R-210 presented in lung cancer tissues and sputum.But other studies also showed a suppressor of mi R-210 in certain cancers by inhibiting cell proliferation and cell cycle,as well as promoting cell apoptosis.Thus we cannot conclude the exact role of mi R-210 in lung cancer progression so far.mi R-630 is another mi RNA still not to be well studied in certain cancer progression.Studies have shown that by acting as a potential modulator of the CIS-response,mi R-630 can regulate CIS-induced cancer cell death in NSCLC and does cytoprotective effect in CIS-administered A549 cells.Dramatically,mi R-630 is proven to play dual roles in cell apoptosis of human lung cancer A549 cells.By recognizing that mi R-630 is a key gene affecting the effectiveness of lung cancer drug therapy,it is extremely necessary for us to explore the function of mi R-630 in lung cancer progression.Transcriptional co-activator with PDZ-binding motif(TAZ),also known as WW-domain-containing transcription regulator 1(WWTR1),acts as a WWdomain-containing transcription co-activator that activates many transcriptional factors.TAZ has been proven to modulate mesenchymal stem cell differentiation,self-renewal of embryonic stem cell,crosstalk with other signaling pathways,and mechano-transduction by binding a series of transcription factors.Furthermore,TAZ plays a crucial role in regulating cell proliferation,apoptosis,tumor formation and organ size in mammals.Previous studies have demonstrated that TAZ is an oncogene in NSCLC and plays an important role in tumorigenesis of lung cancer.Several studies showed that TAZ enhanced lung cancer cell proliferation and tumor development in vivo,and that the high level of TAZ expression correlated closely with poor differentiation,shorter survival and metastasis in lung cancer patients.Objective 1.Whether Up-expression of mi R-210-3p or mi R-630 in lung cancer cell lines can inhibit proliferation of a lung cancer cell.2.Whether Up-expression of mi R-210-3p or mi R-630 in lung cancer cell lines can effect lung cancer cell cancer cell apoptosis or expression of Caspase-3.3.Predicted target gene of mi R-210-3p and mi R-630 by Target Scan,then,the target gene is further selection by a double luciferase reporter element analyzes.4.Whether Up-expression of mi R-210-3p or mi R-630 in lung cancer cell lines can lead to TAZ expression down-regulated.5.Whether Up-expression of mi R-210-3p or mi R-630 in lung cancer cell lines can impact expression of TAZ upstream regulatory genes MST or LATS.Methods 1.Cell line selection: Human lung cancer cell lines included lung aquamous cell line NCI-H2170 and lung adenocarcinoma cell line A549,NCI-H1299,Hcc827.And 293 T for double luciferase reporter analysis.2.Transfected mi R-210-3p mimic or mi R-630 mimic into human lung cancer cell lines and MTT assay for cell growth.The apoptosis of cells was detected by Annexin-V-FITC apoptosis and the expression of Caspase-3 was detected by western blot.3.Identificated TAZ is the target gene of mi R-210-3p or mi R-630: Target Scan predicted and double luciferase reporter test concluded that TAZ was the target gene of mi R-210-3p and mi R-630.Then,the expression of TAZwas detected by q RT-PCR and western blot after transfection of mi R-210-3p or mi R-630 into human lung cancer cell lines.4.Identificated whether mi R-210-3p or mi R-630 can effect expression of TAZ upsteam gene MST or LATS: the expression of MST or LATS was detected by q RT-PCR and western blot after transfection of mi R-210-3p or mi R-630 into human lung cancer cell lines.Results 1.Cell growth was inhibited after transfected mi R-210-3p mimic or mi R-630 mimic into lung cancer cell lines.2.Cell apoptosis and the expression of apoptotic factors Caspase-3 were improved after transfected mi R-210-3p mimic or mi R-630 mimic into lung cancer cell lines.3.Target Scan and double luciferase reporter test concluded that TAZ is common target gene of mi R-210-3p and mi R-630.4.TAZ expression level was significantly reduced after transfected mi R-210-3p mimic or mi R-630 mimic into lung cancer cell lines.5.the expression of MST or LAST can be improved more or less after transfected mi R-210-3p mimic or mi R-630 mimic into lung cancer cell lines.Conclusions 1.Mi R-210-3p and mi R-630 can be tumor suppressor by inhibiting the growth of cells and promoting cell apoptosis in lung cancer cell lines.2.Tumor suppressor mechanism of mi R-210-3p and mi R-630 can promote cell proliferation and inhibition of apoptosis by down-regulating TAZ expression and suppressing TAZ functions.mi R-210-3p and mi R-630 significantly up-regulated the expression of apoptosis factor Caspase-3,and promoted the apoptosis of lung cancer cells.3.Tough mi R-210-3p and mi R-630 interact directly on TAZ,rather than MST and LATS,over-expression of mi R-210-3p or mi R-630 more or less up-regulated MST and LATS expression.Part II:Identification of a novel transcript isoform of the TTLL12 gene in human lung cancerBackground Tubulin tyrosine ligase like 12(TTLL12)is the least characterized and the most divergent member of the TTLL family,which possesses catalytic functions in tubulin post-translational modifications.However,TTLL12 is said to be a pseudo-enzyme having a phylogenetically conserved association of two nonfunctional domains including a SET-like domain and TTL-like domain,for which several differences exist in the structure as compared with other members of the TTLL family.These two domains are associated with histone methylation and tubulin modification,respectively.Recent studies have shown that TTLL12 could affect histone methyla-tion,tubulin modification,mitotic duration and chromosome ploidy in human larynx cancer cells.In addition,there are numerous studies revealing that the TTLL family is closely linked to human cancers,such as neuroblastomas.For example,the TTLL family is often suppressed in human cancers and is positively correlated with a poor prognosis in breast cancer.In prostate cancer,the level of TTLL12 expression was found to be increased in the proliferating layer of benign tissue and during cancer progression to metastasis.These findings suggest that TTLL12 may display the same pattern in other types of cancers,and it could play a crucial role in tumorigenesis and tumor progression.Objective 1.Designed TTLL12 gene 3’RACE primers and amplified the full-length of TTLL12,then found a TTLL12 novel transcript isoform by gene sequencing and bioinformatics software online NCBI BLAST alignment analysis.2.To detected whether the TTLL12 novel transcript isoform exsited in other type of cancer cell line by PCR,gene sequencing and NCBI BLAST comparison analysis.3.The difference between the primary structure and secondary structure of TTLL12 wide gene and TTLL12 novel transcript isoform was analyzed by ioinformatics method,and the function of TTLL12 novel transcript isoform was predicted meanwhile.Methods 1.Total RNA of lung cancer cell line H1299 was extracted as template and the full-length of TTLL12 gene was amplified by Takara 3’ RACE Kit.The full-length was seguenced and NCBI BLAST.2.After the TTLL12 novel transcript isoform was found,two new pairs of TTLL12 primers were designed.Then,to detect whether other cancer cell lines contained TTLL12 novel transcript isoform by PCR,gene sequencing and NCBI BLAST.3.The differences of primary structure between TTLL12 wide-type gene and TTLL12 novel transcript isoform were predicted by bioimformation software Prot Param and Prot Scale.4.The differences of secondary structure between TTLL12 wide-type gene and TTLL12 novel transcript isoform were predicted by bioimformation software DNASta Proteam和 CFSSP.5.The function of TTLL12 novel transcript isoform were predicted by bioimformation software Dis Port.Results 1.A new transcript of TTLL12 gene was found in lung cancer cell line H1299.2.A nucleotide sequence of 108 bp of TTLL12 novel transcript isoform was inserted into the positon between the base 902 to 903 of the coding sequence(CDS)of the TTLL12 wild-type gene.3.The expression of the new transcript of TTLL12 gene was found in all detected tumor cell lines and the three lung cancer cell lines(H1299,H2170 and Hcc827)showed the highest expression levels.4.There were differences of the primary structure and secondary structure between TTLL12 novel transcript isoform and wide-type amino acid chains.5.The additional 36 amino acid chain of TTLL12 novel transcript isoform may give TTLL12 new functionality by being part of a disordered area.Conclusions 1.This study discovered a TTLL12 novel transcript isoform,and a additional nucleotide sequence of 108 bp of TTLL12 novel transcript isoform was inserted into the positon between the base 902 to 903 of the coding sequence(CDS)of the TTLL12 wild-type gene.2.The TTLL12 novel transcript isoform was found to exist in several types of tumor cell line,especially lung cancer cell lines.3.Further study of the use of bioinformatics analysis software to predict that there was a difference between TTLL12 novel transcript isoform and wide-type encoded proteins.4.Further study of the use of bioinformatics analysis software to predict that this additional insert of the nucleotide sequence of TTLL12 novel transcript isoform belongs to the disorder region,suggesting that the additional insert of the nucleotide sequence has apotential biogical function.