| Background and objectiveWith the statistics of more than 1.5 million people dead per year, lung cancer has already been the leading cause among mortality caused by cancer in the world. At present, most of the patients with lung cancer diagnosed at advanced stage. There are some factors that are responsible for poor prognosis and high mortality of lung cancer,such as the atypical clinical characters, difficulty in early diagnosis, difference of histological subtype, lack of understanding in tumor biological characteristics etc.Although new molecular-target durgs for the treatment of certain types of lung cancer has shown a very good prospect, but there is still no suitable targeted therapies for large-scale lung cancer. Overall, the overall survival of lung cancer is still not optimistic, many patients can only survival for a few months after a clear diagnosis.Therefore, it is very crucial to find new diagnostic-related tumor markers or new therapeutic strategies for lung cancer.miRNA is a non coding small RNA composed of 19 to 25 nucleotides, which plays an important role in the regulation of the translation or degradation of the target m RNA in both animals and plants. Mi RNA is one of the important components of numerous genes in multicellular organisms, and it is likely to affect the output of many protein coding genes. miRNA can be divided into oncogenic miRNA and tumor suppressor miRNA in accordance with its regulatory role in the growth anddevelopment of tumor, regulating cell proliferation, invasion, apoptosis and angiogenesis of tumor. The expression pattern of miRNA may be related to the clinicopathological features of certain lung cancer subtypes, suggesting the potential of miRNA as a biomarker in tumor biogenesis, histological type, invasive ability and chemotherapic sensitivity. In general, the miRNA is widly involved in lung cancer,some miRNAs such as let-7 family, miR-34, miR-200, miR-126 and other miRNAs has been widely studied to survey their roles in lung cancer. We have found that miR-339 plays a negative regulated role on growth and invasion in thyroid carcinoma,lung cancer, ovarian cancer, gastric cancer and other tumors through reviewing relevant articles and searching miRBase database. The specific mechanisms of miR-339 to regulate the biological behavior in tumors has not been clarified, in addition, few study has been published in the expression level of miR-339 in lung cancer, especially in lung cancer tissues.At present, most researchers believe growth of tumor depends on angiogenesis to provide sufficient oxygen and nutrients, so the anti-angiogenesis therapy has become a hot spot for the clinical treatment of cancer.VEGF(vascular endothelial growth factor) has been clarified to targeted on vascular endothelial cells and induce new angiogenesis in vivo. Thus, mechanism of inhibiting VEGF has also become the basis for the inhibition of tumor growth. When VEGF combines with VEGF specific receptor, a series of biological effects may occurs.VEGF can promote the neovascularization through inducing the proliferation of vascular endothelial cells; increase the vascular permeability; change the expression of vascular endothelial cells, which are totally conducive to the formation of new blood vessels. This study predicted target gene of miR-339 through three kind of miRNA target gene prediction databases, finally, we screened out VEGF as the target gene miR-339.This research was divided into three parts: the first part analysed the expression level of miR-339 and VEGF in 41 cases of lung adenocarcinoma tissues by qRT-PCR and Western blot; the second part established the stable high expression of miR-339 in A549 lung adenocarcinoma cell lines by the lentiviral vector, investigated the effects of miR-339 upgraduation on the proliferation, invasion, chemotherapy sensitivity and tissue angiogenesis in A549 cell lines and xenograft in nude mice;thethird part further discussed the regulatory mechanism of miR-339 on VEGF after transcription by dual luciferase reporter assay and restore test.Part One Expression level of miR-339 and VEGF in 41 lung adenocarcinoma casesMethods1. 41 cases of lung adenocarcinoma tissue and the corresponding paired adjacent normal tissues were collected.2. The expression levels of miR-339 and VEGFm RNA were detected in 41 specimens by qRT-PCR technique. The correlation between miR-339 and VEGFm RNA was analyzed. Western blot was used to detect the expression of VEGF protein in 41 specimens.3. Expression difference of miR-339 and VEGFmRNA was analysed in different age, sex, lymph node metastasis, degree of differentiation, TNM stage.4. Statistical methods: Statistics data were analyzed using SPSS 21 software, data with normal distribution were discribed in the( x ąs); one-way ANOVA was used for comparison with more than two groups with the prerequisite of normality and homogeneity of variance; LSD-t test was used to compare 2groups of data among several gropus; t test was used to compare two independent sample measurement data, comparison of paired t test for paired samples of two quantitative data; Spearman correlation analysis was used to analyse the correlation of two groups, test level ? =0.05.Results1. The expression of miR-339 in lung cancer tissue was decreased to approximately1/3 compared with normal tissue, while VEGFm RNA expression was almost 3times of normal tissue, there was a negative correlation between expression level of them(R=-0.419, P =0.006).2. The expression difference of miR-339 and VEGFmRNA in different TNM stages and lymph node metastasis was statistically significant(P < 0.05),while none in different ages, genders and differentiations.Part Two Effects of miR-339 upregulation on proliferation,invasion, chemotherapy sensitivity and angiogenesis in lung adenocarcinoma of A549 cell linesMethods1. Lentivirus vector of miR-339 and non-sense sequence was prepared and packaged then transfected into A549 cell lines to establish a stable high expression level of miR-339 in A549 cell lines, the cells was divided to three groups: miR-339 group, NC group, Blank group.2. The expression levels of miR-339 and VEGFm RNA in three groups of cells were detected by qRT-PCR technique. Western blot was used to detect the expression of VEGF, MMP2, MMP9 protein in three groups.3. CCK-8 assay, clone formation assay was used to detect the proliferation of cells in three groups.4. The invasion ability of cells was detected by transwell assay.5. CCK-8 assay was used to detect the proliferation of cells in three groups under different concentrations of cisplatin.6. The xenograft in nude mice model was established, the proliferation of mices in three groups was detected by small animal imaging technology in vivo.7. The proliferation of mices under the treatment of cisplatin was detected by small animal imaging technology in vivo.8. Microvessel density of xenograft in nude mice was detected by immunohisto chemistry technique.9. SPSS 21 statistical software was used for data analysis, test level ?=0.05.Results1. miR-339 and non-sese sequence of recombinant lentivirus vector was established successfully, the two recombinant virus titers were 1.6×108TU/m L and 2.2×108 TU/m L respectively. The expression level of miR-339 in miR-339 group was significantly higher than that in NC and Blank group(P < 0.05).2. The expression level of VEGFm RNA in miR-339 group was significantly lower than that in NC group and Blank group. Westren blot showed that expression of VEGF, MMP2 and MMP9 protein in group miR-339 was significantly lower than that in NC group and Blank group(P < 0.05).3. CCK-8 results showed that the absorbance value of miR-339 group was significantly lower than that of Blank group and NC group in 24 h, 48 h and 72 h,what's more, the difference among miR-339 and other groups showed an increasing trend as time delay(P < 0.05).4. Clone formation assay showed that the cloney number of miR-339 group was significantly less than that of NC group and Blank group(P < 0.05).5. Transwell assay showed that number of penetrating cells in miR-339 group was significantly decreased than NC group and Blank group(P < 0.05), there is no significant difference between Blank and NC groups(P > 0.05).6. CCK-8 was used to detected absorbance values of three groups in 24 hours with different concentration of cisplatin ranging from 0 to 20?g/m L, the results showed that the proliferation activity was decreased following the increasing cisplatin concentration of three groups(P<0.05); the the downward trend of miR-339 group with cisplatin was more pronounced when compared with NC group and Blank group(P<0.05).7. Small animal imaging was used to detect proliferation of xenograft in vivo, the results showed that, fluorescence signal of miR-339 group were significantly decreased compared with Blank group and NC group from the the second week(P<0.05), and with the extension of time, difference of the fluorescence signal in miR-339 group and Blank group or NC group were more pronounced(P<0.05).8. Compared with the Blank group and the NC group, the fluorescence signal of miR-339 group was significantly lower under the treatment of cisplatin(P< 0.05),the fluorescence signal of three groups was all decreased than those in the non-cisplatin group(P<0.05). Among them, the fluorescence signal was decreased more significant in miR-339 group than that of Blank group and NC group under the treatment of cisplatin(P<0.05).9. The microvessel density of miR-339 group was significantly lower than that of Blank group and NC group(P<0.05).Part Three Study on the post transcriptional regulation mechanism of miR-339 on VEGFMethods 1. The target gene of miR-339 was predicted by the target gene prediction database of miRNA.2. Pmir GLO-Wt and pmir GLO-Mt luciferase reporter gene vector was constructed and transfected into A549 cells. Dual reporter gene was used to validate target gene of miR-339.3. PcDNA3.1-VEGF expression vector without 3'UTR VEGF was constructed,pc DNA3.1-VEGF and miR-339 mimics was transfected alone and co-transfected into A549 cells respectively. Western blot was used to detect VEGF expression,the invasion ability of cells was detected by Transwell chamber, restore assay was used to analyze the regulation mechanism of miR-339 on VEGF.4. SPSS 21 statistical software was used for data analysis, test level ?=0.05.Results1. The database predicted that VEGF may be a target gene for miRNA, the complementary pairing area, so-called "seed region" was found between them.pmir GLO-Wt and pmir GLO-Mt reporter gene vector was constructed and transfected into A549 cells successfully.2. Dual luciferase reporter assay showed that luciferase activity of miR-339mimics/pmir GLO-Wt co-transfected group decreased significantly when comparing with miR- 339 scramble/ pmir GLO- Wt and pmir GLO- Mt/ miR- 339 mimics cotransfected Group(P<0.05),.It could be seen that miR-339 may combine with the VEGF 3'UTR region and exert the function of negative regulation.3. pc DNA3.1-VEGF expression vector without 3'UTR was constructed successfully.Restore assay showed that,when transfecting pc DNA3.1-VEGF recombinant vector into A549 cells,both the inhibitory effect of miR-339 in VEGF protein expression and the inhibition of miR-339 on the invasion of A549 cells was reversed.Conclusion1. The expression level of miR-339 downgraduates, while the expression level of VEGF upgraduates in lung adenocarcinoma tissues, the both are negative correlated; Difference exists in the expression level of miR-339 and VEGF in cases of different lymph node metastasis and TNM stage.2. In vitro, upregulation of miR-339 expression can reduce the proliferation and invasion of lung adenocarcinoma A549 cells, increase the sensitivity of cells to cisplatin; animal experiments showed that overexpression of miR-339 can inhibit the proliferation of nude mice, increase its sensitivity to cisplatin, reduced tumor microvessel density.3. VEGF was negatively regulated by miR-339 through targeting the 3 'UTR region of VEGF. |
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