Relativity Between The Expression Of Let-7f In Glioma Cells And Clinical Features And The Effect Of Regulating Target Gene RAB40C On Glioma Cell Proliferation

BackgroundHuman brain glioma is the most common intracranial primary tumor in the central nervous system, and it takes up 60% of all intracranial tumors. The tumor grows dendrically and cannot be removed thoroughly, ending with poor treatment and high mortality. Besides removal by operation, the present treatment is combined with such comprehensive measures as radiotherapy and chemotherapy, resulting in a slight reduction of mortality. However, due to the high ability of invasion and proliferation of glioma, the overall prognosis is not ideal. Mortality in five years exceeds 95%. The treatment of glioma has always been a difficult problem for neurosurgeons in their treatment of tumors in the nervous system.Generally considered glioma is a polygenic abnormal disease. The occurrence and development of glioma mainly come from the activation of proto-oncogenes and the inactivation of anti-oncogenes and the subsequent series of gene abnormality and dynamic evolution. Therefore, the application of molecular biology technique and immunohistochemical technique to test the gene and protein level change will impose a large impact on the research, diagnosis and treatment of glioma. miRNA is conservative in evolution and is a small non-coding RNA molecule (containing about 18-25 nucleotides). It can be combined with the specificity of 3’-untranslated region, resulting in the degradation of target mRNA or the inhibition of its translation, thus regulating genes after transcription. About 1/3 of human genes are regulated by miRNA. Research has found that miRNA participates in the occurrence and development of various cancers, playing the role of "cancer gene" or "anti-cancer gene". There is a large amount of miRNA expression in glioma cells and the miRNA participates in the occurrence and development of glioma cells.There are various kinds of miRNA and let-7 family is one of the most widely researched miRNAs. There are 13 members in let-7 family and Let-7f is one of them, locating at 9q22.3. The present research has found that Let-7f has different expressions in different cancers. Its expression is downregulated in such cancers as ovarian cancer, lung cancer, gastric cancer whereas its expression is upregulated in breast cancer and liver cancer. The abnormal expression of Let-7f may play an important role in the occurrence and development of malignant tumors. At present, the research of glioma is at the preliminary stage. The effect of Let-7f on the proliferation, metastasis and invasion of glioma cells has been reported in research, but Let-7f has many target genes and they form a complicated regulating grid. Different target genes participate in the regulation of tumor cells by Let-7f through different signal pathways. Therefore, the specific mechanism needs to be researched further.miRNA exists not only in brain tissues but also in serum and cerebrospinal fluid samples. miRNA is expressed stably in body fluids and can be tested conveniently, with a high sensitivity and small injury, so it is the ideal biomarker for cancer diagnosis. Research has been documented about the application of blood miRNA in early glioma diagnosis, such as miRNA-205, miRNA-128, and miRNA-210, etc. These biomarkers have different sensitivity and specificity and they have many disadvantages. Until now there does not exist such a miRNA stably tested in serum that is universally accepted by scholars both at home and abroad and can be used in the early treatment and prognostic evaluation of glioma.Not much research has been done on the clinical significance and prognostic value of Let-7f in solid tumors. In order to observe the expression of Let-7f in glioma patients and to explore the role of early diagnosis and prognosis, we tested the Let-7f difference between the glioma patients’tumor tissue and normal brain tissue and between the patients’serum and normal serum. Then we made use of the clinical data, histopathological features, and postoperative survival time, etc. and analyzed the value of Let-7f in the prognostic evaluation and early treatment of glioma. In order to explore the mechanism underlying Let-7f in regulating glioma cell proliferation, we tested the expression difference of Let-7f in glioma cell lines and normal stellate cell lines and the effect of Let-7f on the proliferation ability of in vitro glioma cell growth. We found and verified that RAB40C is the target gene of Let-7f in glioma cells and made a preliminary exploration into the regulatory mechanism of RAB40C in cell proliferation with a view to providing a theoretical basis for the clinical value of Let-7f expression and molecular targeted therapy of glioma. This research is divided into the following two parts.Part 1:Expression of Let-7f in gliomas and its relationship with clinicopathology.Objective:To test the expression of Let-7f in the tumor tissue and serum of glioma patients, to explore the relation between Let-7f expression and patients’ clinical features, and also to explore the value of Let-7f for prognostic evaluation and early diagnosis.Method:1. Real-time fluorescent quantitative PCR (qRT-PCR) technology is used to test Let-7f expression in tumor tissues and peritumoral non-tumor tissues of 108 cases of glioma patients and to test Let-7f expression in the serum of 108 cases of glioma patients and 40 cases of healthy control group patients.2. Relativity is analyzed between Let-7f expression in tumor tissue of glioma patients and their gender, age, tumor size, tumor location, pathologic grade, and KPS score before operation, etc.3. Kaplan-Meier survival analysis is used to study the relativity between the low Let-7f expression in 101 cases of glioma patients with complete follow-up materials and the patients’ survival time after operation4. Single factor and multi-factor Cox proportional hazard regression model is used to analyze the gender, age, tumor size, tumor location, pathologic grade, KPS score, Let-7f expression of glioma patients and to seek for the independent risk factors that affect the prognosis of glioma patients.5. Serum Let-7f expression difference is analyzed in different grades and different groups of glioma patients. ROC curve and its Area Under Curve (AUC) are used to evaluate the early diagnosis value of Let-7f for glioma of different pathologic grades.Result:1.Let-7f expression is low in both glioma tissue and blood.In the tumor tissue samples of 108 cases of glioma patients Let-7f expression is obviously lower than in normal brain tissue (0.45±0.203 VS 1.09±0.211), with significant statistical difference (P=0.000). Let-7f expression in the serum of glioma patients is obviously lower than in the serum of the control group of 40 cases (0.39±0.201 VS 0.88±0.260 P<0.001). There is a relativity between Let-7f expression in the tumor tissue of glioma patients and the corresponding serum samples, with the coefficient R2=0.853 (P=0.01)2. Let-7f expression in glioma tissues is significantly related to tumor size, WHO grading.Let-7f expression in glioma tissues is significantly different in patients of different tumor size and of different pathologic grading, but it has no significant relativity with other factors. All patients in the research were divided into high-level and low-level group around the average expression (0.51) of Let-7f.70.6% of the Ⅲ～Ⅳ patients according to WHO grading were put in the low-level group. In the Ⅰ～Ⅰ patients, only 35.1% has a Let-7f expression lower than the average, the difference is significant (x2=13.588 P=0.000).There is a significant difference in the Let-7f expression between different WHO grading groups (0.33±0.191 VS 0.79±0.20 P<0.001). The low expression rate of Let-7f and the average expression level of Let-7f in patients with tumor diameter above 3cm are significantly different with those with tumor diameter less than 3cm (x2=8.306 P=0.004;0.38±0.193 VS 0.69±0.198 P<0.01).3. The lower the Let-7f expression is in glioma tissues, the shorter the postoperative survival time is.Kaplan-Meier survival analysis shows that among the 101 cases of glioma patients with complete follow-up materials, those with low Let-7f expression in tumor tissue have a shorter postoperative survival time than those with a high Let-7f expression (24.1 month VS 34.5 month). Log rank testing shows the two groups are significantly different (P=0.000). Among the 47 cases of Ⅲ-Ⅳ patients on WHO grading, those with low Let-7f expression have a significantly different postoperative median survival time from those with a high Let-7f expression (13.2month VS 24.5 month P<0.001). However, among the 54 cases of I-II patients on WHO grading, those with low Let-7f expression do not have a significantly different postoperative median survival time from those with a high Let-7f expression (37.3month VS 41.7 month P>0.05).4. Let-7f is the independent risk factor that affects glioma patients’ prognosis.Single factor analysis shows that KPS scores <80, higher grading on WHO and low Let-7f expression in tumor tissue are the factors that affect glioma patients’ prognosis. Univariate and multivariate Cox proportional hazard regression model shows that KPS scores, WHO grading and Let-7f expression in tumor tissue are the independent factors that affect glioma patients’ prognosis. Low KPS scores, high WHO grading and low Let-7f expression in tumor tissues cause poor prognosis. It is showed that Let-7f is significantly related to prognosis and it is the independent risk factor that affects glioma patients’ prognosis (HR=1.971,95% CI=1.381-2.813, P=0.000)5. Serum Let-7f expression has a certain significance for early diagnosis of malignant gliomaSerum Let-7f expression of glioma patients is closely related to WHO grading (P<0.001). Glioma of all grades has a AUC of 0.719 (95% CI,0.653-0.828). The critical value of serum Let-7f is 1.50, and the corresponding sensitivity and specificity are 53.6% and 82.1% respectively. The AUC of high-grade glioma is 0.795 (95% CI, 0.688-0.901), showing that Let-7f has a certain significance for early diagnosis of malignant glioma.Conclusion:Let-7f expression is low in tumor tissue and blood of glioma patients. Let-7f can be a new biomarker for tumor, thus providing help for the evaluation of malignance degree of glioma and its prognosis and giving implication for the early diagnosis of malignant glioma.Part 2:The mechanism underlying the inhibition of glioma cell proliferation by Let-7f through target gene RAB40CObjective:To test Let-7f expression in human glioma cell lines and stellate cell lines, to seek for and verify the downstream target gene of Let-7f in glioma cells, and to explore the mechanism underlying the regulation of glioma proliferation by Let-7f through target gene.Method:1. RT-qPCR is used to test Let-7f expression in glioma cell lines U251, SHG-44, A172, U138, T98G and normal stellate cell lines.2. Transient transfection technique Lipofectamine2000 is used to transfect Let-7finimics, Let-7fmhibitor into A172, T98G respectively. qRT-PCR is used to test transfection rate. CCK-8 is used for cell proliferation experiment, and the proliferation ability of cells were tested at 24h,48h,72h and 96h of cell growth after transfection. Flow cytometry is used to test the growth cycle of cells.3. Databases such as microRNAs、Targetscan and miRBase were searched for the target gene of Let-7f. RT-PCR and Western blot were used to test cell transfection and to study the effect of inhibited and over-expressed Let-7f on target gene transfection and protein expression. Dual luciferase report system is used to verify whether the target gene RAB40C we have sought is the downstream target gene of Let-7f.4. Let-7f and target gene RAB40C were inhibited and over-expressed respectively, and four groups were formed:Group A with control, siRNA-Let-7f, si-RAB40C, siRNA-Let-7f+si-RAB40C; Group B with control, Let-7fmimics, pEGFP-Cl-RAB40C, Let-7fmimics+pEGFP-C1-RAB40C; Group C with control, siRNA-Let-7f, pEGFP-C1-RAB40C, siRNA-Let-7f+ pEGFP-C1-RAB40C; Group D with control, Let-7fmimics, si-RAB40C, Let-7fmimics+si-RAB40C. CCK-8 is used to test proliferation ability of transfected cells. The molecular action mechanisms of Let-7f and its target gene RAB40C in glioma cell proliferation were compared and analyzed.Result:1. Let-7f expression was downregulated in glioma cell lines.Let-7f expression in glioma cell lines U251, SHG-44, A172, U138, T98G is obviously lower than in normal stellate cells (P<0.01). A172 and T98G with the lowest expression were chosen for further experiment.2. Let-7f inhibited glioma cell proliferatioin.Transient transfection method Lipofectamine2000 was used to transfect Let-7fmimics and Let-7finhibitor into glioma cells A172 and T98G. Transfection rate found by qRT-PCR was satisfactory. Cell proliferation ability was reduced after transfected with Let-7fmimics as compared to the group transfected with Let-7finimicsNC or the group untransfected. Flow cytometry test shows that the proportion of cells at G1 and GO phase increased and cells at S phase decreased. The difference is statistically significant (P<0.01).3. RAB40C is the functional target gene of Let-7fAfter searching in the databases microRNA, Targetscan and miRBase for the target genes of Let-7f,it is found that there is a potential binding site between 3’UTR of RAB40C and Let-7f. The result of glioma cells transfected with Let-7fmimics and Let-7finhibitor showed that RAB40C expression was obviously downregulated in Let-7fmimics group as compared to Let-7fmimicsNC group and the group untransfected (P<0.001). Dual-Luciferase reporter assay of gene activity shows that after cotransfected with wild type RAB40C expression vector and Let-7fmimics the relative luciferase activity deceased obviously whereas after cotransfected with wild type RAB40C expression vector and Let-7finhibitor the relative luciferase activity inceased (P<0.01). In the test the relative luciferase activity did not change in cotransfection system of mutant RAB40C with Let-7fmimics and Let-7finhibitor. 4. RAB40C participated in the regulation of glioma cell proliferation by Let-7fIt is found that proliferation ability of siRNA-Let-7f and pEGFP-C1-RAB40C cells increased (P<0.01), proliferation ability of Let-7fmimics and si-RAB40C cells decreased (P<0.01), proliferation ability of siRNA-Let-7f+si-RAB40C and let-7mimicsf+pEGFP-Cl-RAB40C cells did not change obviously, proliferation ability of siRNA-Let-7f+ pEGFP-C1-RAB40C increased obviously (P<0.001), proliferation ability of Let-7fmimics+si-RAB40C decreased obviously (P<0.001). It is found that the inhibition of glioma cell proliferation may be antagonized by over-expressed RAB40C. It is verified that Let-7f through its specificity carries out negative regulation on RAB40C. It decreases the transcription and protein expression of RAB40C, thus inhibiting glioma cell proliferation.Conclusion:RAB40C is the functional target gene of Let-7f. Let-7f carries out negative regulation on RAB40C to inhibit glioma cell proliferation. The regulation of Let-7f and its target gene RAB40C may provide an effective way for glioma treatment.