| Background: Lung cancer is one of the most common malignancies with the highest morbidity and mortality worldwide. Surgical resection was considered the main treatment clinically, while systemic chemotherapy, radiotherapy, neoadjuvant therapy and targeted therapy for post-operation and/or for the patients who were unable to surgery remained the mainly comprehensive treatment models. This comprehensive treatment has improved the prognosis for lung cancer patients. But the 5-year survival rate of the advanced lung cancer patients only around 5%. On the other hand, seeking better treatment models are very important to improve the prognosis for lung cancer patients.Gene splicing is in eukaryotic cells, during the process of the genetic information from DNA to m RNA transcription, removing the introns and juncting the exons together to form the m RNA molecule with continuous coding sequence. In short, the splicing process is to remove introns and retain exons in m RNA precursors(pre-m RNA). Abnormal splicing is in m RNA level, the functional exons or introns happened an exception due to various reasons, which caused the translation abnormal, and eventually the proteins cannot executive its corresponding biological functions. The gene abnormal splicing is closely related to tumor occurrence.Serine/arginine-rich splicing factor 1(SRSF1) is a representative member of the 7 alternative splicing factor protein family. It mainly involved in pre-m RNA splicing and processing, m RNA transport, cell cycle and apoptosis regulation, and other physiological functions. SRSF1 were over-expressed in a variety of tumors. A small portion of scholars suggested that SRSF1 inhibits the nude mice to form tumor in hepatocellular carcinoma cell line and promoted its RNA degradation in nucleus. But most agreed that SRSF1 were a potential proto-oncogene and promoted the development of tumor by alternative splicing. In addition, there is no clear conclusion about the mechanism of SRSF1 related in lung cancer.In our subject, firstly, we used bioinformatics analysis to select the genes interact with SRSF1 gene. And analysis the functions as well as signaling pathways of these genes which were involved. Secondly, detected the expression of SRSF1 gene in lung cancer cell lines and lung cancer tissue. And preliminary discussed the correlations between SRSF1 and clinical pathological characteristics as well as the prognosis in lung cancer. Thirdly, under the gene transfection technology, we studied the biology behavior and the possible mechanism that the SRSF1 gene affected on lung cancer cell. Lastly, in order to provide a possible direction for the therapeutic program of lung cancer in the future, we discussed the sensitivity of antitumor treatment that the SRSF1 gene related in lung cancer cell.Objective:1、In order to sure the relationship between the expression of SRSF1 gene and lung cancer clinically, we detected the expression of SRSF1 gene in lung cancer cell lines and lung cancer tissues. And preliminary discussed the correlations between SRSF1 and clinical pathological characteristics as well as the prognosis in lung cancer.2、In order to lay a foundation for further study, we studied the biology behavior and the possible mechanism that the SRSF1 gene effected on lung cancer cell.3、In order to provide a possible direction for the therapeutic progam of lung cancer in the future, we explored the sensitivity of antitumor treatment that SRSF1 related in lung cancer cell.Methods:8 1、Using the c Bio Portal database to select the genes interact with SRSF1 and pick out the genes with Pearson value > 0.4. Utilizing STRING analysis to draw the protein network diagram with these gene expression products. Then classified and analyzed the role and function of these genes with Gene Ontology analysis and KEGG pathway analysis.2、In lung cancer cell lines, we detected the expression of SRSF1 in eight kinds of lung cancer cell lines and two kinds of bronchial epithelial cell lines both in protein level and RNA level by Western blot and q-PCR technology. In lung cancer tussues, we selected seven paired lung cancer tissues and normal lung tissue from clinical lung cancer patients to detect the expression of SRSF1 in protein level utilizing Western blot method. In order to further detect the expression of SRSF1, we used the immunohistochemical technique to expand the sample size in 60 paired lung cancer tissue microarray. We also preliminary discussed the correlations between SRSF1 and clinical pathological characteristics as well as the prognosis in lung cancer.3、We constructed six kinds of plasmids included p CDH, p LKO.1, SRSF1, SRSF1-si RNA1, SRSF1-si RNA2, SRSF1-si RNA3 and designed the forward and reverse primers with the related enzyme cutting site respectively. Using the PCR technology to colon the whole length of target gene, the agarose gel electrophoresis to recycle the PCR products, then under the processing of enzyme cutting, purification, connection and transformation in Escherichia coli. Then we identified and screened the positive colony PCR products in use of corresponding antibiotics and extracted the plasmids in Escherichia coli, recycled and DNA sequenced. We used the right sequence of the plasmids to transfect to virus after detecting the normal expression in protein level. We used HEK-293 T cell to package the purpose plasmids with auxiliary plasmids and virus particles, and collected the virus supernatant to infect the target NCI-H1299 cell and screened stable cell lines.The expression of SRSF1 gene in stable cell lines was detected by western blot and q-PCR technique. Finally we studied the impact of the biological functions and possible mechanism through colony formation, wound healing, transwell migration and invasion models, flow cytometry, animal model experiments after SRSF1 9 over-expression and/or down-expression in lung cancer cell.4、In order to discuss the sensitivity of antitumor treatment that the SRSF1 gene related in lung cancer cell, we chose four kinds lung cancer cell lines p CDH、SRSF1、p LKO.1 、 SRSF1-si RNA2, and exposed them to chemotherapy, thermotherapy, radiotherapy, thermo-chemotherapy and thermo-radiotherapy. By cisplatin(DDP) sensitivity experiments, we calculated the cell lines with IC20 value and IC50 value of DDP. Though CCK-8 experiment and flow cytometry technology to detect proliferation and apoptosis effect on lung cancer cell line with different expression levels of SRSF1 after anti-tumor treatment.Results:1、There were 2678 genes correspond to SRSF1 which were enriched in the cell cycle, DNA metabolism, response to DNA damage repair, DNA replication, cellular response to stress, RNA splicing and other functional areas. These proteins are mainly involved in cell cycle, spliceosome formation, ubiquitin protein degradation mediated nonsense, RNA degradation, p53 and TGF-? signal pathway.2、SRSF1 protein was highly expressed in NCI-H1299, NCI-H446, NCI-H2170 and NCI-H520 cell lines. And its m RNA was highly expressed in NCI-H1299, NCI-H446, NCI-H358 and NCI-H520 cell lines. SRSF1 protein was highly expressed in seven lung cancer tissues compared to normal lung tissues. In tissue microarray, the immunohistochemical results showed in sixty paired tissues, the positive expression rate of SRSF1 in lung cancer tissue(95%) was obviously higher than in normal lung tissues(13.33%). This result had remarkable statistical significant(P<0.0001).3、The SRSF1 expression level was positively correlated with tumor size, T stage, N stage and clinical stage(p<0.05), while there had no significance with age, gender and pathological types(p>0.05). In SRSF1(-/+) group and(++) group, the 1 year, 3 years, 5 years survival rates were 93.90%, 72.09%, 59.30% and 64.20%, 24.10% and 4.02% respectively. The results in two groups had remarkable statistical difference(P<0.0001).4、The Western blot and q-PCR results showed the protein and m RNA expression levels were increased after SRSF1 over-expressed in NCI-H1299 cell line, while they decreased after SRSF1 silenced in NCI-H1299 cell line. The silence results were better in SRSF1-si RNA1 and SRSF1-si RNA2 group than SRSF1-si RNA3 group.5、Over-expression SRSF1 gene could promote the proliferation, migration, invasion ability in lung cancer cell. It also promoted tumor formation in nude mice, suppressed apoptosis and change the cell cycle. The colony formation experiments showed, colony formation rate in SRSF1 group(63.93%±2.01%)was higher than Mock group(43.14%±1.94%) and Control group(43.41%±0.85%). And the results had statistical difference(P<0.01).The wound healing experiments showed, the cells in SRSF1 group moved quicker than Control group and Mock group both in 6th hour and 16 th hour(P<0.05). The transwell migration experiments showed the number of cells that through the chamber was more in SRSF1 group(334.6±18.4)compared to Control group(214.4±17.4) and Mock group(217.2±13.8), the results had statistical difference(P<0.01). The transwell invasion experiments showed the number of cells that through the chamber was more in SRSF1 group(367.6±17.4)compared to Control group(236.4±13.4) and Mock group(251.6±12.6), the results had statistical difference(P<0.01). According to the results showed by flow cytometry technology, over-expression SRSF1 gene could suppress apoptosis in lung cancer cell line, the rate in SRSF1 group(2.8%±0.3%)was less than in Control group(5.8%±0.7%), but the results had no statistical difference yet(P>0.05). Over-expression SRSF1 gene changed the cell cycle, the cell proportion at the G0/G1 phase was decreased from 51.43%±0.21%(in Control group) to 38.05%±4.47%(in SRSF1 group), the G1/G2 phase was decreased from from 14.09%±0.38%(in Control group) to 1.32%±0.62%(in SRSF1 group), while the S phase increased from 34.49%±0.59%( in Control group) to 62.0%±4.47%(in SRSF1 group), all of the change had statistical difference(p<0.05). Over-expression SRSF1 gene also promoted tumor formation in nude mice. The tumor formation rate in SRSF1 group(87.50%)was higher than Control group(62.50%). The tumor weight in SRSF1 group(1650.60±1200 mg) was heavier than Control group(328.42±249.18 mg). The results in two groups had remarkable statistical difference(p<0.001)。11 6、When silenced SRSF1 gene, it could inhibit the proliferation, migration and invasion ability in lung cancer cell. The colony formation experiments showed, colony formation rate in si RNA1 group(14.21%±0.93%)and in si RNA2 group(26.89%±2.13%)were both lower than Mock group(52.43%±1.83%) and Control group(53.35%±2.15%). The results had statistical difference(P<0.01). The wound healing experiments showed, the cells in si RNA1 group and si RNA2 group moved quicker than Control group and Mock group both in 6th hour and 20 th hour(si RNA1 group P<0.01, si RNA2 group P<0.05). The transwell migration experiments showed the number of cells that through the chamber was less in si RNA1 group(21.4±10.4)and in si RNA2 group(86.2±19.2)than Mock group(344.4±30.4)and Control group(343±18), the results had statistical difference(P<0.01). The transwell invasion experiments showed the number of cells that through the chamber was less in si RNA1 group( 196.4±20.4) and in si RNA2 group( 295.2±21.2) than Mock group(507.4±20.4)and Control group(508.4±19.4), the results had statistical difference(P<0.01).7、Over-expression SRSF1 gene, the expression of N-cadherin and Snail was increased while the expression of E-cadherin was decreased. Silencing SRSF1 gene, the expression of N-cadherin and Snail was decreased while the expression of E-cadherin was increased.8、The sensitivity of the lung cancer cell against tumor treatment was reduced after SRSF1 gene over-expression. According to DDP sensitivity experiments, the IC20 value was 9.21±2.13 ?g/ml and the IC50 value was 24.82±4.02 ?g/ml in SRSF1 group which was higher than the IC20 value(6.71±3.16 ?g/ml)and IC50 value(14.09±2.76 ?g/ml) in p CDH group, the results had statistical difference(P<0.05). CCK-8 experiment showed that the anti-tumor treatment on SRSF1 group had weaker inhibition on cell proliferation compared to p CDH group. Thermotherapy combined chemotherapy or radiotherapy had better inhibit effect on cell proliferation than single treatment. The flow cytometry experiment confirmed that after anti-tumor treatment, the apoptosis rate in SRSF1 group was less than p CDH group. The results in radiotherapy group and thermotherapy combined groups had statistical difference(P<0.05). Cell apoptosis rate in thermotherapy combined groups was increased compared to single treatment groups.9、The sensitivity of the lung cancer cell against tumor treatment was increased after silenced SRSF1 gene. According to DDP sensitivity experiment, the IC20 value was 2.88±1.23 ?g/ml and the IC50 value was 6.62±1.15 ?g/ml in si SRSF1 group which was lower than the IC20 value(6.13±2.47 ?g/ml)and IC50 value(13.90±2.30 ?g/ml)in p LKO.1 group, the results had statistical difference(P<0.01). CCK-8 experiment showed that the anti-tumor treatment on si SRSF1 group had greater inhibition on cell proliferation compared to p LKO.1 group. Thermotherapy combined chemotherapy or radiotherapy had better inhibit effect on cell proliferation than single treatment. The flow cytometry experiment confirmed that after anti-tumor treatment, the apoptosis rate in si SRSF1 group was more than in p LKO.1 group, and all of the results had statistical difference(P<0.05). Cell apoptosis rate in thermotherapy combined groups was increased compared to single treatment groups.Conclusion:1、The expression of SRSF1 gene was much higher in lung cancer, and it positively correlated with the tumor growth and lymph node metastasis in lung cancer.2、The high expression level of SRSF1 gene was an important risk factor to affect the prognosis of lung cancer patients, which might become a potential indicator to evaluate the effect of prognosis and monitoring efficacy in lung cancer.3、SRSF1 gene promoted malignant biological behavior in lung cancer cell.4、SRSF1 gene might be positively regulate the expression of the transcription factor Snail to control the occurrence of EMT in lung cancer cell.5、The expression level of SRSF1 gene was proved as a negative factor correlated with the sensitivity of anti-tumor treatment in lung cancer cell.6 、 Thermotherapy could enhance the sensitivity of chemotherapy and radiotherapy in lung cancer cell.7、Downregulation SRSF1 gene may become a new direction and target for lung cancer treatment in the future. |
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