| Lung cancer, the leading cancer-related cause of death, is the most dangerous cancer to human health and life worldwide. In recent years, the death rate of lung cancer was obviously increased and became the the fastest growth rate and leading cancer-related cause of death in China. The traditional method of lung cancer treatment is usually based on the histopathological types of lung cancer, which mainly divided into two types, namely small cell lung cancer and non-small cell lung cancer(NSCLC). NSCLC consists of squamous-cell carcinoma, large cell carcinoma, and adenocarcinoma. Lung adenocarcinoma is the most common histological subtypes of lung cancer, accounting for70%of global lung cancer. The best treatment for NSCLC is surgical resection. Postoperative5-year survival rate for NSCLC patients in stage I can reach73%. Sadly, most lung cancer is diagnosed too late for curative treatment. Although great progresses have been made in understanding the disease, the therapeutic effect not gained significant improvement and the5-year survival rate was only16%. Metastasis was the leading cause of treatment failure and death of patients. Previous studies demonstrated that epithelial-to-mesenchymal transition was the important process of distant metastasis of cancer. Deeply investigating the role of EMT in the development of lung cancer was the key for lung cancer prevention and remedy.Functional proteins are considered as the executor of the functional gene, mRNA is the intermediay. With the completion of the human genome project, the genome sequence encoding protein only accounts for2%of the entire genome sequence, but the rest of98%of genome sequence encodes a lot of the noncoding RNA. Nc-RNAs, especially small Nc-RNAs, such as microRNAs, make the gene regulation into RNA era.MiRNAs are a class of small RNAs (21-24nt) that regulate the expression of target genes at the post-transcriptional level by base pairing with the3’untranslated regions (UTRs) of mRNAs and promoting mRNA unstability. They are first transcribed from miRNA genes in the genome as primary miRNAs (pri-miRNAs) which are processed into the mature miRNAs through two sequential cleavage steps and exported from the nuclei to cytoplasm. Firstly, depending on the complementarity between miRNA and3’untranslated region (UTR) of target mRNA there are three known mechanisms of miRNAs action on mRNAs:1) target mRNA degradation when miRNA is near-perfectly complementary with target mRNA,2) translational inhibition with little or no influence on mRNA levels when miRNA is only partially complementary to its target mRNA and3) the above two ways coexist. Application of bioinformatics method can predict the same target genes exist in multiple microRNA binding site based on the above mechanism and characteristics of the sequence of microRNA5’2-8bits nucleotide should fully complementary to the target mRNA3’UTR, indicating regulation of microRNA target gene expression possibly through diverse ways. In addition to the negative regulation function, minority microRNA also can positive regulate gene expression. Secondly, it is evident that single miRNAs may regulate translation of numerous downstream mRNAs and each mRNA is likely to be regulated by several miRNAs simultaneously. At last, microRNA regulate gene expression depending on the context of cells and tissues and the physiological and pathological state. It is obvious that the regulatory process of microRNA is complicated. miRNAs are implicated in cell differentiation, proliferation, apoptosis, and various physiologic and pathologic processes, including growth and development. Clinical studies showed that microRNAs were closely related to the occurrence of lung cancer, for example, miR-146b, miR-155, let-7e, miR-34a, miR-34c-4p, miR-25, miR-191were closely related to the prognosis of NSCLC. Defects in the biogenesis of microRNAs are contributed to the development of lung cancer. Dicer is the best-established regulator of miRNA processing. Deletion of Dicer abrogates the production of mature miRNAs. Reduction of Dicer expression levels was correlated with poor survival of NSCLC patients. Specical microRNAs are existed in lung cancer. More than40microRNAs are involvd in the pathogenesis of lung cancer, including let-7a, miR-17-92, miR-34, miR-21, miR-29, miR-155, miR-31, and miR-200et al, they decreased or increased in lung cancer and function as oncogene or anti-oncogene. At present, the representatives of tumor suppressor microRNA in lung cancer are let-7, miR-29and miR-34a.MiR-17-92, a polycistronic microRNA Cluster, comprises six miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a) and resides in intron3of the C13orf25gene at13q31.3. miR-17-92is overexpressed in cancer, such as lung cancer, B-cell lymphomas and liver cancer et al. MiR-17-92promotes the development of cancer by targeting proapoptotic gene Bim, anti-oncogene p21and pten, TGFβ, TGFBR2, Rbl, TSP-1, CTGF and E2F1. MiR-19, a key oncogenic component for miR-17-92, promotes the proliferation of cancer cells by suppressing the expression of pten and Bim.MiR-17-92family members play an important role in lung cancer. Supression of miR-17-5p and miR-20a can lead lung cancer cells overexpressing miR-17-92to apoptosis. Thus, it is valuable for studying the effect of miR-19on the development of lung adenocarcinoma.To further investigate the effect and the potential mechanism of miR-19on the lung adenocarcinoma cell line A549, the following several aspects will be studied:(1) determining the expression level of miR-19in several lung cancer cell lines, observing the effect of miR-19on morphology, proliferation and migration of A549 cells in vivo and vitro;(2) determining the effect of miR-19on gene expression pattern of A549cells;(3) the regulation mechanism of miR-19on A549. The effect and potential mechanism of miR-19on A549will be obtained though above research.The results are as follows:1Effect of miR-19on the morphology, proliferation and migration of A549cells1.1Determining the expression of miR-19in5lung cancer cell line using quantitative RT-PCRThe relative expression quantity are calculated by2-△△Ct. The expression of miR-19are higher in5lung cancer cell lines than that in BEAS-2B. The expression of ml9is the highest in non small cell lung cancer cell line H23, but the lowest in lung adenocarcinoma cell line A549and A549-Luc-CM-1. The statistics of One Way Anova showed that the expression of miR-19were significantly different in6lung cell line(F=797.865, P<0.001). Using2-△△Ct method, compared with BEAS-2B cell line, miR-19increased1.941,1.541,2.458,2.696,4.419-fold in A549, A549-luc, H446, H460and H23, respectively.1.2Generation of lentivirus vector of pm19H2BmRFP, pm19(-)H2BmRFP1.2.1Amplification of the sequence of miR-19a/19b; miR-19a(-)19b(-)Amplification of the sequence of miR-19a/19b (784bp); miR-19a(-)19b(-)(981bp) based on MIG19a/19b; MIG19a(-)19b(-) plasmid. The PCR products were electrophoresed in1%agarose gel, and distinct bands were appeared at784bp and981bp, respectively.1.2.2Identification the recombinant vector by PCRThe PCR products of miR-19a/19b, miR-19a(-)19b(-) were cut by Enzymatic digestion and purified, ligated, transformed, then colonies were selected to be identified. The result showed that the target fragment located at784bp,981bp were amplified from eight pm19H2BmRFP colonies and nine pm19(-)H2BmRFP colonies.1.2.3Identificating sequence of the recombinant vectorThe sequence of the recombinant vectors are in accordance with MIG19a/19b; MIG19a(-)19b(-), indicating the success of generation of pm19H2BmRFP and pm19(-)H2BmRFP.1.2.4Lentivirus package and titer determination of control vector and overexpression vectors of pm19H2BmRFP and pm19(-)H2BmRFPCotransfection293T cells with three plasmid package system (Control pHIV-H2BmRFP or pm19H2BmRFP or pm19(-)H2BmRFP, psPAX2and pMD2.G) produced three lentiviruses, including pHIV-H2BmRFP, pm19H2BmRFP and pm19(-)H2BmRFP.48h later, red fluorescence were observed in the nucleus of293T cells. Assaying virus titers by gradually diluting the virus to infect293T cells. The titers of virus of pHIV-H2BmRFP, pm19H2BmRFP and pm19(-)H2BmRFP are6.1x106TU/ml,5.2x106TU/ml and5.0x106TU/ml, respectively.1.3Generation of lung cancer cell line overexpressing miR-19a/19b and miR-19a(-)19b(-)The lentivirus of control PHIV-H2BmRFP, pm19H2BmRFP and pml9(-)H2BmRFP, encoding miR-19a/19b, miR-19a(-)19b(-) were used to infect A549-Luc cells, respectively.48h later, red fluorescence were observed in the nucleus of293T cells.1.4Determining the miR-17, miR-18a, miR-19, miR-20a and miR-92a in A549-Luc cells overexpressing miR-19, miR-19(-) using quantitative PCR.Compared with A549-Luc, miR-19increased1.893times in A549/RFP+/m19; miR-17, miR-20a, miR-92a increased0.839,6.938and1.546times respectively; miR-18a are not obviously changed in A549/RFP+/m19(-) cell.1.5Effect of miR19a/19b and miR19a(-)19b(-) on the morphology of A549-Luc cellSeveral passages later, the morphology of A549cells overexpressing miR-19a/19b and miR-19a(-)19b(-) changed. Compared with A549/RFP+/H2B cells, the morphology of A549/RFP+/m19showed elongation, spindle-like, fibroblast shape, sporadic distribution; A549/RFP+/m19(-) showed cell gibbosity, clump together.1.6Effect of miR-19a/19b, miR-19a(-)19b(-) on the proliferation of A549cells.Determining the ability of proliferation of A549/RFP+/m19, A549/RFP+/m19(-) and A549/RFP+/H2B using MTT assay. Single repeated measure ANOVA showed that the ability of proliferation was significantly different in three groups. Compare to A549/RFP+/H2B, the speed of proliferation of A549/RFP+/m19and A549/RFP+/m19(-) cells slowed down(P<0.001).Flat colony formation assay experiments also showed that the ability of proliferation was significantly different in three groups(F=76.155, P<0.001). The above mentioned experiments showed overexpression of miR-19and miR-19(-) suppressed the proliferation of A549-Luc cell in vitro.1.7Effect of overexpression of miR-19a/19b and miR-19a(-)19b(-) on cell cycle of A549-LucThe cell cycle of A549-Luc overexpressing miR-19a/19b and miR-19a(-)19b(-) were determined using FACS. Compare to A549/RFP+/H2B, the ratio of G1phase in A549/RFP+/m19(P=0.011) and A549/RFP+/m19(-)(P<0.001) were significantly upregulated, the ratio of S phase in A549/RFP+/m19(P=0.001) and A549/RFP+/m19(-)(P<0.001) were significantly downregulated. This result showed that A549/RFP+/ml9and A549/RFP+/ml9(-) cells were blocked in G1phase.1.8Overexpression of miR-19a/19b, miR-19a(-)19b(-) on the migration of A549cells.Detection the migration ability of A549/RFP+/H2B, A549/RFP+/ml9and A549/RFP+/ml9(-) cells using Transwell chamber showed that the migration ability was different in three groups (F=57.284, P<0.001). Compared with A549/RFP+/H2B cells, the migration ability of A549/RFP+/m19cells obviously increased(P<0.01), but in A549/RFP+/m19(-) cells decreased significantly(P<0.01).1.9Determined the expression of E-cadherin and Vimentin using quantitative RT-PCRCompared with A549/RFP+/H2B, the expression of E-cadherin decreased in A549/RFP+/m19cell line(P<0.05), and increased in A549/RFP+/m19(-)(P<0.05), respectively; the expression of Vimentin increased in A549/RFP+/m19cell line(P<0.05), and decreased in A549/RFP+/m19(-)(P<0.05).1.10Determined the expression of E-cadherin and Vimentin using Western blotCompared with A549/RFP+/H2B, the expression of E-cadherin and Vimentin were downregulated and upregulated in A549/RFP+/m19cell line, respectively; the expression of E-cadherein was not significantly downregulated in A549/RFP+/ml9cell line, but the expression of Vimentin decreased in A549/RFP+/m19(-).1.11In vivo optical imaging of subcutaneous xenografts of A549/RFP+/H2B and A549/RFP+/m19cellsFor subcutaneous transplantation of A549/RFP+/H2B cells, the photons of bioluminescence had significantly increased over time(F=17.037, P<0.001), For subcutaneous transplantation of A549/RFP+/m19cells, we found that the photons of bioluminescence had also increased over time(F=3.850, P<0.024), but the proliferation speed significantly lowered than that of A549/RFP+/H2B cells(P<0.001).2Effect of miR-19on the gene expression pattern of A549cells2.1Data analysis on the gene expression pattern47,000genes were determined. Cluster analysis showed significant difference of gene expression between A549/RFP+/ml9and A549/RFP+/H2B cells. Compared with control vector group,586genes were up expressed and504genes were down expressed in A549cell with overexpression of miR-19(A549/RFP+/m19).Using MAS data analysis system derived from Boao corporation, GO was used to analyze the differentially expressed genes between A549/RFP+/ml9and A549/RFP+/H2B cells. The result showed that GO Term were significantly enriched in Signal transduction, Cell adhesion, Transcription, Oxidation reduction and EMT. Compared with A549/RFP+/H2B cells, the marker of EMT, such as CDH2(N-cadherin), CDH11(OB-cadherin), ITGA5, ITGB6, COL3A1, COL1A1, COL5A1, FN1, CALD1, MMP1, MMP2and MMP9were upregulated, while CDHl(E-cadherin) and KRT19were downregulated in A549/RFP+/H2B cells. Furthermore, differentially expressed genes related to EMT were analyzed by GO functional classification. The result showed that these gene were involved in the process of physiogenesis, metabolism and physiology regulation. Pathway analysis showed these genes had multiple signal pathway between each other. 2.2Expression of E-Cadherin, N-cadherin, Vimentin, Fibronectin, snail, MMP1and MMP10was examined in A549/RFP+/H2B and A549/RFP+/ml9using quantitative PCR.Compared with A549/RFP+/H2B, E-cadherin expression decreased in A549/RFP+/m19, while the expression of N-cadherin, Vimentin, FN1, Snail, MMP1and MMP10significantly increased in A549/RFP+/m19.2.3Expression of E-Cadherin, Vimentin, Fibronectin, snail, MMP1and MMP10were detected in A549/RFP+/H2B and A549/RFP+/m19using western blot.Compared with A549/RFP+/H2B, E-cadherin expression was obviously downregulated in A549/RFP+/m19. Vimentin expression was slightly upregulated, while expression of Fibronectin、snail、MMP1and MMP10were obviously upregulated in A549/RFP+/m19.3Preliminary study on the molecular mechanism of induction EMT of A549by miR-19The expression of Pten、STAT3、p-STAT3、ITGA5. P53and c-myc was detected in A549/RFP+/H2B cells and A549/RFP+/m19cells using western blot. Compared with A549/RFP+/H2B, Pten, P53and c-myc were downexpressed, while STAT3, P-STAT3and ITGA5was upexpressed in A549/RFP+/m19cells.From the above3sections experiments, we can conclude that:(1) miR-19can induce the transformation of human lung adenocarcinoma cell line A549to the mesenchymal cell.(2) miR-19promotes the transformation of A549cells to mesenchymal cells perhaps via suppression of Pten, following activation of STAT3and Snail, and suppression of E-cadherin, upregulation of Vimentin and ITGA5. |
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