| BackgroundLung cancer is one of the fastest-growing cases of malignant tumors, with world leading incidence and mortality rates. It is a serious threat to human health and lives. On January 25,2016, CA:A Cancer Journal for Clinicians, the global top journals in the field of cancer research, has published online the Cancer Statistics in China,2015 released by the National Cancer Center of China. The Statistics summarized that an approximated 4,291,600 new cancer cases would be diagnosed and 2,814,200 cancer-related deaths would occur in China in 2015; while lung cancer would be the leading common incident cancer and the top causes of cancer-related deaths among other types of cancer. According to the report, there are about 733,300 new lung cancer cases, and 610,200 lung cancer-related deaths in China in 2015. Although there are significant and considerable improvement in the both diagnosis and therapeutic approaches of lung cancer, it still has a poorer 5-year survival rate, which is less than 15%.The poor survival rate could be attributed to the following reasons. On one hand, the current understanding of the mechanism of lung cancer’s development, progression, and metastasis is still limited and unsure; in addition, the effective and sensitive early-stage diagnosis methods and monitoring indicators are still scarce, which lead to most patients have been diagnosed as lung cancer at its advanced stage. This is one of the major reasons for the poor prognosis of the lung cancer patient. On the other hand, except surgery approach on lung cancer, the conventional radiotherapy and chemotherapy is still provably ineffective. As the progress with the clinical application of the targeted therapy, this lung cancer treatment effect has notable improvement. However, there is also an urgent need to solve the problems emerged from this application, such as how to improve the effectiveness of targeted therapy, and how to solve the problem of drug resistance during the treatment, etc.Previous studies indicated that abnormalities of gene expression and gene regulation might be the reasons and roots of cancer’s development and progression. Through the in-depth study on the mechanism of lung cancer, we can do the effort to locate the possible existed biomarkers related to the biological activity of lung cancer, and explore its function on the mechanism of lung cancer, which hope we will improve the treatment of lung cancer eventually.In the early cancer research, researchers were majorly focusing on the study of the impact of the protein-coding gene on the pathogenesis of lung cancer. According to the Human Genome Research conducted at the end of last century, in the human genome transcript process, only 2% of the genes have the protein encoding capability, and more than 90% of the genes do not have the protein encoding capability, which would be transcribed into the non-coding RNA (ncRNA). These ncRNAs have been widely considered that they play a significant regulation role in the development and progression of lung cancer. Based on the length of nucleotides, ncRNA can be categorized in two types:short non-coding RNA (sncRNA), which length of nucleotides is shorter than 200nt, and long non-coding RNA (lncRNA), which length of nucleotides is longer than 200nt. In a relative long period of time, researchers are focusing more on studying sncRNA, and finds that sncRNA plays an important role in the mutual regulation between it and its host genes during the tumors’development stage. IncRNA was once considered as a by-product of the transcription of RNA polymerase II (RNAP â…¡) and did not have any biological function; and it was the "noise" and/or "garbage" of the genes transcription. As the further researches on IncRNA in the recent years, lncRNA has been found that it can influence the tumor’s development through multiple mechanisms; and it has been also identified playing similar important role during this progression as sncRNA.Based on the point of view from current researches, lncRNA involved in the development and progression of a variety of tumors, including breast cancer, liver cancer, prostate cancer, lung cancer, melanoma, and etc. Further studies also show that IncRNA can achieve its regulatory function on gene expression at the transcriptional, post-transcriptional, and epigenetic level.According to previous researches on the relationship between IncRNA and lung cancer, it demonstrated that some lncRNA might be involved in the processes of lung cancer cell proliferation, invasion, metastasis, apoptosis, and drug resistance, and etc. In order to better understand the relationship between lncRNA and tumors, we have collected 83 clinical specimens of primary non-small cell lung cancer and cancer-adjacent tissues between January 2014 to June 2014 at the Department of Thoracic Surgery, General Hospital of Guangzhou Military Command of P.L.A. Based on the microarray screening approach, we have found some lncRNA differences between the lung cancer tissue and cancer-adjacent tissues. Among those, we have selected the lncRNA DB327252, which has a relative higher differentiation between the cancer tissues and cancer-adjacent tissue, as the subject of this research. In this research, we try to further understand the effect of lncRNA DB327252 on the mechanism of development and progression in Lung Cancer.Methods1. Collection of the lung cancer tissue specimensDuring the period from January 2014 to June 2014, lung primary cancer tissue specimens and cancer-adjacent tissues were collected from the patients underwent the radical surgery of lung cancer or palliative resection of lung cancer at the Department of Thoracic Surgery, General Hospital of Guangzhou Military Command of P.L.A. The cancer-adjacent tissues must be at least 5 centimeter apart from the edge of the cancer tissue. We have collected 83 specimens, where patients were all diagnosed as primary pulmonary non-small cell lung cancer, and were not treated by preoperative radiotherapy, chemotherapy, targeted therapy, and immunotherapy. Meanwhile, the general clinical information and detailed pathological records has also been collected. The collection of these biological specimens was consistent with the aseptic principles, and was approved by the patients and the Ethics Committee of the General Hospital of Guangzhou Military Command of P.L.A.2. Cell lines and Cultivation methodThe normal human bronchial epithelial cell lines, BEAS-2B and 16HBE-N, were obtained by procurement or donation; lung cancer cell line A549, H1299, H446, and 16HBE-T cell line induced by malignant transformation, were cultivated in MEM (Modified Eagle’s Medium, Hyclone, USA) or RPMI-1640 (Invitrogen, Carlsbad, CA, USA), with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-streptomycin mixture (100IU/ml penicillin, and 100μg/ml streptomycin), where mediums were put in the humidified incubator with temperature at 37℃ and 5%(v/v) CO2.3. Total RNA Extraction and qRT-PCR analysisTotal RNA was extracted from cell by the application of Trizol methodology. We performed microarray experiments using the fluorescence dye (SYBR Green â…¡) method, detected lncRNA, and identified the lncRNA DB327252 expression in each cell line based on the real-time quantitative analysis (qRT-PCR), and finally selected the cell lines for the further experimental study.4. Transient transfection of siRNA interference sequence:After prepared the lncRNA DB327252 siRNA/Lipofectamin-e-2000 complex, A549 and 16HBE-T cells were transfected using the complex following the manufacturer’s instruction. Quantitative analysis of the interference efficiency was performed by the qRT/PRC methodology. And the most efficient siRNAs (>70%) were selected for the further research. During these processes, the most appropriate concentration and the best time for transfection treatment for processing research-used siRNA were also explored and tested.5. Cell Proliferation assayThe appropriated amount of cells was seeded in the 96-well plates. The cell proliferation was evaluated by using CCK-8 kit (Cell Counting Kit-8) at 6h,12h,24h, 48h, and 72h respectively after the transfection process. The sample absorbance value has been identified by the Automatic Microplate Reader; and the cell viability has been calculated based on the cell proliferation rate formula.6. Colony formation assayLogarithmic growth phase cells were harvested and seeded in the 6-well plates. After cultivation for 14 days, the experiment was terminated once visible cell clones on the plates. The cell colonies were stained with 0.1% crystal violet staining, and then colony formation rate was calculated.7. Cell cycle assayAfter the appropriate amount of cells were seeded in the 6-well plates, it was placed and cultured in aseptic antibiotics medium with temperature at 37℃ and 5% CO2 environment for 24 hours; and then transfection of siRNA was performed. After 48 hours of the siRNA transfection, cells were harvested and fixed with 70%ethanol, put in-20℃ environment for overnight; and then they were stained with Propidium Iodide (PI). Finally, the cell cycle distribution was analyzed by the flow cytometry.8. Cell apoptosis assayAfter the appropriate amount of cells were seeded in the 6-well plates, it was placed and cultured in aseptic antibiotics medium with temperature at 37℃ and 5% CO2 environment for 24 hours; and then transfection of siRNA was performed. After 48 hours of the siRNA transfection, cells were harvested, washed, fixed, and permeabilized. Cells were stained in accordance with the instructed steps of Annexin V-FITC/PI kits; and then the cell apoptosis was analyzed by the flow cytometry9. In vitro cell invasion assayAfter the appropriate amount of cells were seeded in the 6-well plates, it was placed and cultured in aseptic antibiotics medium with temperature at 37℃ and 5% CO2 environment for 24 hours; and then transfection of siRNA was performed. After 24 hours of the siRNA transfection, the cells were digested by trypsin and generated a single cell suspension. A density of 5x105 cells per well were seeded in the 24-well plates upper chamber of the Transwell application; and then add 500μl DMEM (Dulbecco’s Modified Eagle Medium), which contain 20% of the FBS, into the lower chamber. After 24 hours’culture, the rate of cell invasion was calculated.10. Cell healing assay (Scratch assay)After the appropriate amount of cells were seeded in the 6-well plates, it was placed and cultured in aseptic antibiotics medium with temperature at 37℃ and 5% CO2 environment for 24 hours; and then transfection of siRNA was performed. After 24 hours of the siRNA transfection, until the cells were grown to a confluent cell monolayer, a pipet tip was used to create scratches (vertical dash) on the cell monolayer. The exfoliated cells were washed off from the plate and replaced with the desired medium to culture for 24 hours with temperature at 37℃. The cells would be observed and photographed under a microscope every 6 hours; and the cell growth coverage area ratio was calculated.11. soft agar colony formation assay6-well plate was laid 2ml 0.6% of agar as the bottom layer; and then a 2 ml cell mixture (cell concentration is 1×104 cells/ml) containing 0.3% low melting soft agarose was added to each well, which became the top agar. The plate was cultured in the incubator for 21 days and then the number of clones (which is bigger than 50 cells) was counted.12. Establish stable transfected cell linesThe shRNA was transfected with the selected medium with the concentration of 500 μg/mL G418 and Lipofectamine-2000. After 24 hours of transfection, cells were split to 1:4. After cultured for 10 to 14 days, the isolated (individual) monoclonal appeared, which was generated to a single cell suspension and counted. The culture of cell was continued until the large number of cell amplification appeared, when the total RNA was extracted. The protein expression of each clone obtained was detected to verify whether the expression level was reduced by quantitative real-time PCR (qRT-PCR), which was used to identify the stable transfected cell lines.13. Nude mice Tumorigenicity assayCells, which harvested and digested by trypsin, were washed by phosphate-buffered saline (PBS) twice and were resuspended. A 150μl of PBS with approximately 5×106 suspended cells were injected into the inguinal subcutaneous of each nude mice. The mental state, reaction, diet, motility, hair color, hair smoothness, and the tumor growth at the inguinal subcutaneous of each nude mice was observed and recorded as the tumor latency diary. The long diameter (a) and short diameter (b) were measured every other day once the tumor was formed. After 28 days, the nude mouse was scarified and the tumors were removed and weighed.14. Statistical AnalysisSPSS 20.0 was used for the statistical analysis. All experimental data were repeatedly measured at least three times, and the data value was expressed as mean ± S.D. (x±s). Statistically significant difference analysis between two data groups was using two-sided t-test, while multiple data groups’comparison (more than two groups) was using single-factor variance analysis (one-way ANOVA). It is considered the difference is statistically significant if the p-value is less than 0.05 (p<0.05).Results1. Microarray result analysisMicroarray screening approach has been applied to the RNA extracted from the 83 clinical specimens of primary non-small cell lung cancer and cancer-adjacent tissues. The expression level of lncRNA DB327252 in the cancer tissue is higher than in the cancer-adjacent tissue; and this result is statistically significant as p is less than 0.05 (p<0.05).2. The relationship between the expression of lncRNA DB327252 and clinical characteristic of lung cancerBased on the result, the high expression level of lncRNA DB327252 is not related to patient’s gender, age, and lymph node metastasis as p is bigger than 0.05 (p>0.05), which is not statistically significant. On the other hand, the high expression level of lncRNA DB327252 is related to smoking (p=0.045), histological type (p=0.023), size of tumor (p=0.037), and TNM stage (p=0.002), where p-value shows that these results are statistically significant. Moreover, the results also indicate that the expression level of smoker is higher than non-smoker, the expression level of adenocarcinoma is higher than non-adenocarcinoma, the expression level of tumor group which size is bigger or equal to 3 centimetres is bigger that the tumor size smaller than 3 centimeters, and the expression level of TNM stage â…¡ to â…£ is significantly higher than TNM stage â… .3. The expression level of lncRNA DB327252 in different cell linesCompared to the normal BEAS-2B cell line, the expression level in 16HEB-N is (1.184±0.137) times, which is not statistically significant as the p is bigger than 0.05; the expression level in 16HBE-T, A549, H1299, H446, and H460 is (16.259±1.209), (20.596±1.81), (17.009±0.890), (16.088 ±2.002), and (12.496 ± 0.907) times compared to the expression level in BEAS-2B cell line respectively; and these results are all statistically significant as the p-value are less than 0.05 (p<0.05).4. Screening of siRNA sequencesIn order to downregulate the expression level of targeted gene, a normal control sequence and four siRNA sequences were used respectively for transfection. The results found that in the A549 cell line, the efficiency of interference of siRNA-2, siRNA-3, and siRNA-4 is 66%,77%, and 58% respectively; and this result is statistically significant as p<0.05; In the 16HEB-T cell line, the efficiency of interference of siRNA-2 and siRNA-3 is 65% and 67% respectively; and this result is statistically significant asp<0.05;5. The impact of lncRNA DB327252 on cell proliferationIn the A549 cell line, compared to blank control group, the cell survival ratio of siRNC-nc group is (99.15±2.87)%, which is not statistically significant as p-value is bigger than 0.05; compared to the siRNA-nc group, the cell survival ratio of siRNA-2 group and siRNA-3 group is (36.20±1.88)% and (34.17±2.27)% respectively, and the result is statistically significant as p<0.05; In the 16HEB-T cell line, compared to blank control group, the cell survival ratio of siRNC-nc group is (96.21±1.20)%, which is not statistically significant as p>0.05; compared to the siRNA-nc group, the cell survival ratio of siRNA-2 group and siRNA-3 group is (37.32±5.41)% and (39.00±1.72)% respectively, and the result is statistically significant as p<0.05.These results show that when the expression level of lncRNA DB327252 was downregulated, cell proliferation was inhibited.6. The effect of lncRNA DB327252 on Colony formationCompared to A549/siRNA-nc, the colony formation rate of A549/siRNA-1 and A549/siRNA-2 is not statistically significant as p>0.05; Compared to 16HEB-T/siRNA-nc, the colony formation rate of 16HEB-T/siRNA-1 and 16HEB-T/siRNA-2 is not statistically significant asp>0.05.7. The impact of lncRNA DB327252 on cell cycleIn the A549 cell line, the ratio of G0/G1 cells in the siRNA-2 group increased to 80.7%, and this difference is statistically significant (p<0.05); the ratio of G0/G1 cells in the siRNA-3 group increased to 81.3%, and this difference is statistically significant (p<0.05); while in the G2/M and S phase, the ratio is not statistically significant different as p>0.05. In the 16HEB-T cell line, the ratio of G0/G1 cells in the siRNA-2 group increased to 84.22%, and this difference is statistically significant (p<0.05); the ratio of G0/G1 cells in the siRNA-3 group increased to 83.0%, and this difference is statistically significant (p<0.05); while in the G2/M and S phase, the ratio is not statistically significant different asp>0.05.The above results demonstrate that when the expression level of lncRNA DB327252 was downregulated, the ratio in the GO/GI phase of the cell cycle increased, and the tumor growth was inhibited.8. The effect of lncRNA DB327252 on cell apoptosisCompared to A549/siRNA-nc group, the cell apoptosis ratio of A549/siRNA-1 and A549/siRNA-2 is not statistically significant (p>0.05). Compared to 16HEB-T/siRNA-nc group, the cell apoptosis ratio of 16HEB-T/siRNA-1 and 16HEB-T/siRNA-2 is not statistically significant (p>0.05).9. The effect of lncRNA DB327252 on cell invasion ability24 hours after the transfection processed, compared to A549/siRNA-nc group, the cell invasion of A549/siRNA-1 and A549/siRNA-2 is not statistically significant (p>0.05); Compared to 16HEB-T/siRNA-nc group, the cell invasion of 16HEB-T/siRNA-1 and 16HEB-T/siRNA-2 is not statistically significant (p>0.05).10. The impact of lncRNA DB327252 on cell migrationCompared to A549/siRNA-nc group, the ratio of scratch pitch to the original pitch in A549/siRNA-1 and A549/siRNA-2 is not statistically significant (p>0.05). Compared to 16HEB-T/siRNA-nc group, the ratio of scratch pitch to the original pitch in 16HEB-T/siRNA-1 and 16HEB-T/siRNA-2 is not statistically significant (p>0.05).11. The result of soft agar colony formationIn A549 cell line, compared to the shRNA-nc group with (64.93+5.10)%, the colony formation of shRNA transfection soft agar silent roup is decreased to (23.60±5.05)%, this difference has obviously statistically significant with p-value is less than 0.01; In 16HEB-T cell line, compared to the shRNA-nc group with (67.73±6.56)%, the colony formation of shRNA transfection soft agar silent roup is decreased to (34.80±6.06)%, this difference has obviously statistically significant as p<0.01.12. The effect of lncRNA DB327252 on nude mice TumorigenicityThe growth of tumor in the A459/shRNA stably transfected group is slower than in the shRNA-nc group, where this result is statistically significant (p<0.05); similarly, the growth of tumor in the 16HEB-T/shRNA stably transfected group is also slower than in the shRNA-nc group, it is also statistically significant (p<0.05).The volume of tumor in the A549/shRNA is significantly lower than A549/shRNA-nc group (667.32±41.35 mm3 vs.1691.45±44.70 mm3), this difference is statistically significant (p<0.05). The weight of tumor in the A549/shRNA is significantly lighter than A549/shRNA-nc group (233.3±86.37 mg vs.448.2±120.8 mg), it is statistically significant (p<0.05); The volume of tumor in the 16HEB-T/shRNA is significantly lower than 16HEB-T/shRNA-nc group (633.18±46.75 mm3 vs.1448.20 ± 85.69 mm3), this difference is statistically significant (p<0.05). The weight of tumor in the 16HEB-T/shRNA is significantly lighter than 16HEB-T/shRNA-nc group (267.3±74.86 mg vs.391.4±86.61 mg), the result is statistically significant (p<0.05).The above results show that when the expression level of lncRNA DB327252 was downregulated, the speed of tumor growth, volume and weight of tumor have been reduced in the vivo tumor test. As a result, the growth of tumor has been reduced.Conclusion1. The high expression of lncRNA DB327252 has been observed in the non-small cell lung tissue collected from patients, which provides evidence to support that lncRNA DB327252 has the function of oncogenes. It is also believed that the high expression level is associated with the increasing level of malignancy of the non-small cell lung cancer.2. For the view point of statistics, the expression level of lncRNA DB327252 observed from the collected non-small cell lung tissue is not related to patient’s gender, age, and lymph node metastasis, as they were not statistically significant (p>0.05); while it is related smoking, histological type, size of tumor, and TNM stage, as they all have a p-value less than 0.05, which is considered as statistically significant. The results observed that the expression of lncRNA DB327252 is relatively high in the group of smoker, adenocarcinoma, tumor size bigger 3cm, or TNM stage from â…¡ to â…£.3. The expression of lncRNA DB327252 is relatively high in A549ã€H1299〠H446ã€H460 and 16HBE-T cell lines. It shows that lncRNA DB327252 has the malignant biological activity.4. The results suggest that lncRNA DB327252 promotes the tumor’s transformation process and development majorly through the regulation of cell proliferation on A549 and 16HEB-T, and the cell cycle.5. Based on the results of soft agar colony formation in vitro experiment and nude mice tumorigenicity experiment, it can be confirmed that lncRNA DB327252 play an important role in promoting tumor growth and development. |
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