Function Analysis Of MiRNAs Associated With Breast Cancer Cell Proliferation And Metastasis

MicroRNAs(miRNAs) are a class of approximately 22-nuleotide-long noncoding RNAs which are expressed in many organisms.Mature miRNAs arise from one arm of endogenous hairpin transcripts by sequential processing in the nucleus and cytoplasm.First,miRNAs are transcribed by RNA polymeraseâ…¡as the long primary miRNAs(pri-miRNAs) in the nucleus. Second,the pri-miRNAs are cleaved by the endonuclease Drosba to release the shorter precursor miRNA(pre-miRNA),which are 60- to 70-nucleotide-long imperfect hairpin structure.Finally,pre-miRNAs are exported to cytoplasm by exportin-5 and are processed by the endonuclease DICER to generate the 22-nucleotide RNA duplexes,one strand of which is the mature miRNA,miRNAs regulate target genes post-transcriptionally, by inhibiting the translation through imperfect base-pairing interaction with the 3' -untranslated regions(3' -UTRs) of their respective target genes,or degrading their target mRNAs through perfect or near-perfect base pairing.Recent large-scale studies have revealed that there are about 533 miRNA loci in huamn.The number of miRNAs may be more than 1% of the total protein-coding genes,but about 30%of protein- coding genes are regulated by miRNAs.miRNAs have important roles in many biological processes,such as development,cell death,proliferation,differentiation and disease.And increasing evidences show that mutation and differential expression of some specific miRNAs are associated with the formation and progress of many types of cancers,including brain cancer,chronic lymphocytic leukemia,colorectal neoplasia,hepatocellular carcinoma,lung cancer, lymphomas,papillary thyroid carcinoma,testicular germ cell tumors,etc.Breast cancer is one of the most common cancers in adult females and studies showed that breast cancer was also associated with the deregulation of miRNAs.The expression of miRNAs have been examined in a wide range of breast cancer cell lines,and in clinical normal and cancer breast tissues,but less is knowed about their exact mechanism.We carried out our reseash in function of miRNA associated with breast cancer.Part 1Previous studies showed that the underexpressed miRNAs in cancers, such as let-7,may function as tumor suppressor genes and inhibit cancers by regulating oncogenes and genes that control cell differentiation or apoptosis.In order to investigate the tumor suppressor miRNAs in breast cancer,we focused on the miRNAs whose expression decrease in breast cancer.According to some miRNA databases and the published miRNAs profiles in breast cancer and other types of cancers,we chose 10 miRNA candidates,including mir-30d,99a,100,125b1,130a,126/126*,145,191, 199a and 214.We assayed their effects on cell by overexpressing them.We found that mir-126 could strongly suppress cell growth in HEK293 by inhibiting the cell cycle transition from G1/G0 to S1 phase and had the same effect on breast cancer cell MCF-7,which was in accordance with the recent finding that mir-126 can reduce metastasis of malignant cells(CN34) obtained From the pleural fluid of a patient with metastatic breast cancer,by significantly suppressing growth of tumor volume in mice.We went on to investigate the mechanism of proliferation suppression of mir-126.To identify the mRNA targets of mir-126,we performed a computational screen for genes with mir-126 complementary sites in their 3'-UTR using several open access database.Considering the the suppressing effect of mir-126 on cell cycle progression,5 genes which have a high prediction score and are associated with cell growth were chosen.3'-UTR luciferase report assay suggested that mir-126 negatively regulated IRS-1.Further studies showed that mir-126 suppressed translation of IRS-1 mRNA and had no effects on mRNA level.And knockdown of IRS-1 can also suppress cell growth in HEK293 and breast cnacer cell MCF-7,which recapitulate the effects of mir-126.Quatative assay revealed that expression of IRS-1 protein was negatively correlated with mir-126.In a conclusion,this part of work revealed that mir-126 suppressed cell growth by inhibiting cell cycle transition from G0/G1 to S phase. Considering recent reports,we supposed that mir-126 supressed cell growth by intervening IGF1R-PI3K/Akt signal pathway.Part 2miRNAs are difficult to detect due to their small sizes(about 20 nt). Besides northern blot,most of traditional methods for miRNA detection are based on step-loop PCR.These methods are tedious and costly for high-throughput screening because each miRNA needs one specific revere transcription(RT) reaction.For example,if we used stem-loop RT-PCR to detect 30 miRNAs in 4 cell lines,120 reverse transcription reactions would be needed in addition to 4 reactions for reference gene,which is very sample-demanding.Because there are only 6 bp-complement between miRNA template and its specific RT primer,step-loop PCR often has low specificity and amplification efficiency.So Taqman probes are often used but it is costly.We developed a simple method called poly(A) RT PCR,which has many advantages:1) it is simple.One sample only needs one RT reaction to detect different miRNAs;2) the efficiency of RT reaction is high because of the 13 bp-complement between anchor RT primer and tailed RNA templates;3) it has high specificity because of the additional 12-(dT) distance between the binding sites of miRNA-specific forward primer and the universal reverse primer.With this method,we detected expression of 22 miRNA in breast cancer cell lines with different metastasis potentials.Results showed that mir-29a/c,mir-99a,mir-100 and mir-125b1 were up-regulated in high metastasis breast cancer cell lines and mir-191 was down-regulated in high metastasis breast cancer cell line,which was confirmed by real-time RT-PCR.Except mir-191,the same results were recaptulated in other breast cancer cell lines.And we used universal Taqman probe to quantify the miRNAs and got the results accordant with previous ones.Use of the universal taqman probes made polyA RT-PCR more precise.miRNA inhibitor assay revealed that inhibition of mir-125b can suppress cell growth and reduced the ability of penetrating martrigel in breast cancer cell lines MDA-MB-231 and BT-549.In order to investigate the role of the 3 miRNAs in breast cnacer cell metatasis,we construted the MCF-7 cell lines stably expressing mir-99a,mir-100 and mir-125b1.In this part of our work,we constructed a new and simple method detecting miRNA.With this method,we found 4 miRNA was associated with breast cancer line metatastasis and mir-125b may be involed in cell proliferation an invision.Research of these 4 miRNAs is being procceded and the effects of the 4 miRNAs on metatasis will be determined by animal experiment in vivo.And we will detect their expression in breast cancer tissues with different metatasis.We believe that our results will be meaningful to research of breast cancer miRNAs and miRNA application in diagnosis and treatment of breast cancer.Due to the advantages,polyA RT-PCR will find much application in future.Part 3HOXA10 induces expression of P53 and reduces expression of SNAIL,so HOXA10 acts as tumor suppressor.Previous studies showed that HOXA10 is often silenced by promoter methylation in high metastic breast cancer cells and tissues.But some clinical researches showed that there were no redcued expression of HOXA10 in some high metastic breast cancer samples,and we detected a high expression level of HOXA10 in high metastic breast cancer cell BT-549.So we suspected that,like many other members of HOX gene family,HOXA10 may be regulated at the translation level by miRNAs.By computational screening,we selected two candidate miRNAs-mir-196a and mir-135a.With 3' UTR luciferase assay,we found mir-135a can significant reduced the report gene activity of HOXA10 3' UTR,and this result was confirmed by mutating the predicted mir-135a binding site at HOXA10 3' UTR.And RT-PCR results showed that mir-135a is highly co-expressed with HOXA10 in BT-549.Inhibition of mir-135a reduced cell invision in breast cancer cell BT549. A recent report showed mir-135,as a tumor suppressor,can regulate the Adenomatous Polyposis Coli(APC) gene in colorectal cancer.This part of our work first preliminariely revealed that mir-135a can suppressed HOXA10 and is asscociated with cell invision.We will investigate the function of mir-135a in progress of breast cancer and confirm the regulation of HOXA10 by mir-135a.