Antitumor Effects Of MicroRNA-Let-7a And MicroRNA-34a And Their Molecular Mechanisms In Lung Cancer In Vitro

Lung cancer is the most commonly diagnosed malignancy and the leading causeof mortality among all types of cancers. Every year more than1million deaths arecontributed by lung cancer. Lung cancer can be divided into two main histologicaltypes’ i.e. non-small cell and small cell lung cancer (NSCLC and SCLC respectively)NSCLC accounts for75-85%of lung cancer. MicroRNAs are non-coding, small(~20–22nucleotides long) single-stranded RNA molecules, novel class of generegulators. Inhibit gene expression at transcriptional or translational level. Inmammals, it is now predicted that microRNAs control the activity of more than60%of all protein-coding genes. MicroRNAs take part in regulation of almost everycellular process known so far like growth, development, and apoptosis. Changes inexpression of microRNAs are associated with many human disorders especiallycancers and some microRNAs can inhibit tumor growth through different signalspathways. Recently scientist had innovated microRNA mimics, which are chemicallysynthesized, double stranded RNAs mimics mature endogenous miRNAs aftertransfection into cells. In our study we hypothesized that microRNA let-7a and miR-34a mimics both they can act as tumor suppressor. MicroRNA let-7is highlyconserved in diverse animal species from worms to humans located at a chromosomeregion that is usually deleted in cancer. There are13and14different let-7familymembers in human and mouse respectively, in human these different members areLet-7a-1,a-2,a-3,7b,7c,7d,7e,f7-1,f7-2,7g,7i,mir-98,and mir-202.Let-7a has identicalsequence across various animal. The miR-34family composes of three miRNAsincluding miR-34a, miR-34b and miR-34c that are encoded by two different genes;miR-34a is encoded by its own transcript, found in the region of chromosome1p36.23,and usually it is aberrantly expressed in multiple types of diseases such as B-lymphoidmalignancies, colon, and many other types of diseases. Exogenous over-expression ofmiR-34a in many cancers cell line lead to reactivation of apoptotic pathways, whichrecommends that miR-34a is a tumor suppressor. Working through our study we hadconducted another experiment in order to identify the most suitable reference genesfor gene expression studies using RT-qPCR in lung cancer. Objectives: The field of microRNA is a new phenomenon, the associationmicroRNAs-apoptosis, cell cycle interrelationship is still in infancy, the efficacy ofmicroRNAs that selectively target important signaling pathways involved in thecontrol of lung cancer is obscure. However exploring this filed could help us tounderstand in depth the molecular pathways that control apoptosis, cell cycle andoffer future therapeutic perspectives in lung cancer. In our study we were interested tostudy the anti-tumor effects of microRNA let-7a and microRNA34a mimics in lungcancer in vitro and their mechanisms on apoptosis, cell cycle, and cell growth.Methods:(1) microRNA let-7a and microRNA34a mimics were designed and synthesizedby GenePharma.company (2) Two lung cancer cell lines were enrolled in this study(A549and NCI-H446). Each cell line was divided into four groups, the let-7a group,miR-34a group and negative control group, and non-transfected A549or NCI-H446cell line groups (3) RT-qPCR was used to detect endogenous miRNA expression oflet-7a,miR-34a to confirm their transfection into the cell,48to72hours posttransfection, cells were harvested and subjected to different experiments.(4) Cellproliferation assay was performed in all four groups using MTT (5)) Apoptosis andcell cycle were analyzed by fow cytometry (6) Total RNA was extracted; reversetranscribed into cDNA (7) mRNA expressions of k-ras,CCDK4, bcl-2,c-myc,Bax,P53and p57were carried out by RT-qPCR and analyzed usingcomparative CT method (8) Western blot was used to analyze C-MYC,BCL2andP53protein expression levels (9) In order to identify the most suitable reference genesfor gene expression studies using real-time quantitative PCR in lung cancer, mRNAsexpression encoding a panel of10housekeeping genes (HKGs) including18S,GAPDH, RPLP0,ACTB, PPIA, PGK1, B2M, RPL13A, HPRT1, and TBP wereexamined using real-time quantitative PCR (RT-qPCR) in human lung cancer celllines including A549, NCI-H446, and NCI-H460(10).The Best keeper, norm Finder,and geNorm software were employed to ascertain the most suitable reference genes tonormalize the RNA input.Results:1. Ectopic microRNA let-7a and miR-34a mimics were successfully transfectedinto lung cancer cell line A549and NCI-H446. 2. Cell proliferation was inhibited in both A549and NCI-H446lung cancertransfected with microRNA let-7a and miR-34a mimics at48h posttransfection.3. Apoptosis analysis results displayed that microRNA let-7a and miR-34amimics induced cell apoptosis in both A549and NCI-H446cells48h posttransfection. The apoptosis results in microRNA let-7a, negative control, non-transfected A549groups were10.93±0.642911.4±0.4,1.53±1.02respectively; and microRNA34a,negative control and non-transfected A549groups were13.93±1.40,2.27±0.75and2.03±0.40respectively; while theapoptosis percentage in microRNA let-7a, negative control and non-transfected groups NCI-H446were9.4±1.41,1.4±0.3,2.57±0.37,respectively.And microRNA-34a, negative control and non-transfected NCI-H446groups were12.3±1.72.1±0.62,1.97±0.37, respectively.4. Cell cycle analysis result showed that microRNA let-7a microRNA34ainduced cell arrest at G0/G1phase in both A549and NCI-H446cell lines48hpost transfection. The percentages of cells in the G0/G1phase were75.42±1.34,64.94±1.54, and63.74±1.83in microRNA let-7a, negative controland non-transfected A549and groups respectively. And in microRNA34anegative control, non-transfected A549groups, the G0/G1percentage was76.85±.27,64.94±1.54,63.74±1.83respectively. And the percentage (in mean±SD) of cells in G0/G1phase in microRNA let-7a, negative control and non-transfected NCI-H446cell lines was70.98±.72,59.57±0.8,60.51±0.78respectively. And the G0/G1percentages in microRNA34a, negative controland non-transfected NCI-H446groups were73.34±1.0760.57±1.157,60.11±1.78respectively.5. Relative mRNA expression levels of bcl-2,C-myc and k-ras were significantlylowered and p53,CCDK4and Bax expressions levels were markedly increasedin microRNA let-7a and miR-34a A549and NCI-H446cells than thosecontrol and non-transfected groups.6. Western blot analysis results showed that relative protein expression levels ofbcl-2and C-myc were significantly lowered (P<0.05) in microRNA let-7a andmiR-34a transfected A549and NCI-H446cells than those control groups. P53protein expression level was markedly increased in miR-34a transfected group (P<0.05), compared to negative control group cells of A549and NCI-H446cell.7. in order to identify the most suitable reference genes for gene expressionstudies using real-time quantitative PCR in lung cancer, best keeper softwarerevealed that all10(HKGs) were stable particularly the GADPH followed by18S were the most stable genes whereas PPIA and HPRT1were the leaststable genes. The norm Finder program showed that the PPIA followed byACTB were the most stable while B2M and RPLP0were the least stable. Andthe geNorm program revealed that ACTB and PGK1followed by PPIA werethe most stable genesConclusion:This thesis focus on the tumor inhibitory roles and mechanisms of microRNAlet-7a and miR-34a transfection in A549and NCI-H446lung cancer cell lines,andIdentification of suitable reference genes for gene expression studies using real-timequantitative PCR in lung cancer.1. We successfully transfected microRNA let-7a and miR-34a transfection inA549and NCI-H446lung cancer cell lines respectively.2. The MTT results revealed that ectopic transfection of microRNA let-7a andmiR-34a transfection in lung cancer inhibit growth of both cell lines3. Ectopic transfection of microRNA let-7a and miR-34a mimics’ transfectionin lung cancer induced cell cycle arrest at G0/G1phase, and induced cellapoptosis.4. Expression levels of mRNAs of p53, CCDK4, p57and bax genes weresignificantly up regulated in both lung cancer cell lines A549and NCI-H446transfected with microRNA let-7a and microRNA-34a mimics48hours post transfection while bcl-2, k-ras and c-myc were significantlydown-regulated.5. Protein expression levels of bcl-2and C-myc were significantly lowered(P<0.05) in microRNA let-7a and miR-34a transfected A549and NCI-H446cells groups.P53protein expression was up-regulated in miR-34atransfected groups. 6. Based on the three analyzing programs, best keeper, norm Finder, andgeNorm ACTB, PPIA, and PGK1were identified as the most stablereference genes in RT-qPCR studies lung cancer cell lines.Our work demonstrated that, microRNA let-7a and miR-34a mimics they caninduce cell cycle inhibition and apoptosis. ACTB, PPIA, and PGK1were identifiedfor the first time as the most stable reference genes in normalization of RT-qPCRstudies in lung cancer Moreover further researches are highly recommendedincluding inhibitors of microRNA let-7a and mainly miR-34a and gene knockoutin vivo and vitro to confirm our results.