| Background:In China,malignant tumors have become a serious public health problem.Among them,lung cancer is the malignant tumor with the highest morbidity and mortality in China,and it has been on the rise in recent years.According to China’s annual report on cancer,the overall incidence of lung cancer showed an upward trend from 2000 to 2014.In 2014,the estimated number of new cases of lung cancer was 781,000,and the incidence rate was 57.13/10 million;45.8/100,000,ranking the first cause of death from malignant tumors in the same period.Non-small cell lung cancer(Non-small cell lung cancer,NSCLC)is the most common pathological type in lung cancer.The lack of specific symptoms in the early stage causes about 2/3 of patients to be in the advanced stage at the time of diagnosis and lose the chance of surgery.Although current treatments including chemotherapy,radiotherapy,targeting,and immunotherapy have made great progress,due to the heterogeneity of tumor cells and genomic instability,these treatments can only benefit a small number of patients,and still It is prone to drug resistance and relapse,and the 5-year survival rate has not yet achieved a major breakthrough.At present,the pathogenesis of lung cancer is still not very clear.The lung cancer mutation consortium(LCMC)study found that about 2/3 of advanced lung cancer cases have detected the presence of at least one driver mutation gene3,and the development of drugs targeting these target genes can improve the prognosis of some lung cancer patients.At present,the existing targeted drugs have shown good efficacy against the dominant population of target gene mutations.Therefore,if we can find new driver genes,it may be possible to solve the treatment dilemma of some lung cancer patients.High mobility group(HMG)protein is a kind of rich and highly conserved non-histone chromosomal protein.It is named because of its high mobility during electrophoresis.Members of this protein family exist widely.It is abundant in quantity and can be combined with DNA or nucleosomes to induce changes in chromatin structure.It is of great significance to the process of chromosome dynamics and DNA transcription and translation in chromosomes.HMGs are divided into classical HMG protein and non-classical HMG protein.The classic HMG protein was discovered earlier and can be divided into three subfamilies:HMGA(High mobility group A protein),HMGB(High mobility group box protein)and HMGN(high mobility group nucleosome).The box domain and nucleosome binding domain bind to DNA or nucleosomes and participate in the replication,transcription,translation,and epigenetic modification of chromosomal DNA.The non-classical HMG family members include HMGXB3(HMG-box containing 3),HMG2L1(high mobility group 2 like 1),HMG20A,HMG20B four members,all found late,and the current research is also less.The classic HMG protein has been found to have a certain correlation with lung cancer,especially the most in-depth study of HMGB1 involved in the occurrence,metastasis of lung cancer and even chemotherapeutic drug resistance.Is HMG2L1,which also contains the HMG box domain,in the development of lung cancer?Does it work?Based on this assumption,this study first explored the expression levels of HMG2L1 in cancer tissues and adjacent tissues of lung cancer surgical resection specimens by immunohistochemistry,and found that the expression levels of HMG2L1 in cancer tissues were significantly higher than those in adjacent tissues Then,we further expanded the sample size for testing,and analyzed the relationship between the expression level of HMG2L1 and the clinical characteristics of lung cancer patients;at the same time,we explored the function and possible mechanism of HMG2L1 lung cancer development at the cellular level,using shRNA to interfere with HMG2L1 Expression,and study its effect on lung cancer cell proliferation,migration,invasion,and preliminary exploration of possible mechanisms.Purposes:1.Explore the expression of HMG2L1 in non-small cell lung cancer and its relationship with clinical features and prognosis;2.Explore the role and possible mechanism of HMG2L1 in non-small cell lung cancer.Methods:1.Detection of HMG2L1 expression in lung cancer tissues:Collect paraffin specimens of lung cancer tissues that were surgically removed,detect the expression of HMG2L1 by immunohistochemistry,and adopt the H-score scoring method for semi-quantitative scoring.At the same time,the baseline clinical data of the included cases were collected,including:gender,age,smoking status,comorbid disease,tumor size,lymph node metastasis,distant metastasis,clinical stage,pathological tissue differentiation,diagnosis and treatment,etc.,And follow up regularly.Analyze the correlation between the expression level of HMG2L1 and the clinical data of patients(the statistical method uses chi-square test)and the relationship between HMG2L1 expression and survival prognosis.2.Cell experiment:1.Cell culture:Purchase human NSCLC cell lines A549 and H1299cells from the ATCC cell bank,and cultured with RPMI1640 medium containing 10%serum,penicillin and streptomycin in an incubator at 37°C,5%CO2 and saturated humidity When the cells have grown to 90%confluence,trypsinization and passage,and inoculate into new culture flasks according to the inoculation density of 2×10~5/ml,and subculture each flask every 2-3days.3.Construction of the HMG2L1-shRNA lentiviral vector According to the human HMG2L1 gene sequence,design three pairs of interfering oligo sequences through the website,and Hand over to the company to synthesize and construct plasmid vector,sequence analysis,packaging into lentivirus,and transfecting to in A549 cells,the transfection efficiency was detected by qRT-PCR method,and the best interference sequence virus was selected for amplification to obtain the HMG2L1-shRNA lentiviral vector.4.Transfection of HMG2L1-shRNA lentivirus:Transfect HMG2L1-shRNA lentivirus into A549 cells and H1299 cells,extract protein and RNA of cells 72 hours after transfection,and Western blot and qRT-PCR were introduced to identify transfection efficiency.5.Establishment of stable cell expressing cell lines:A549 cells and H1299 cells transfected with HMG2L1 were added to a medium containing blasticidin that had been pre-explored for screening,and the cells were passaged repeatedly after repeated fluid selection.Western blot and qRT-PCR to identify the expression of HMG2L1;6.Cell function test:1.cell proliferation:A549 cells and H1299 cells transfected with control/HMG2L1-shRNA lentivirus were seeded on a 24-well plate.The cells were stained with crystal violet staining on days 2,4,and 6 after seeding.Select the 595nm wavelength on the immunoassay device to measure the absorbance of each group of cells,draw a growth curve according to the absorbance,and calculate the cell proliferation rate;7 Scratch experiment:A549 cells and H1299 cells transfected with control/HMG2L1-shRNA lentivirus were seeded on 6-well plates,and scratched when the cells grew to 90%confluence.Observe the cell migration every 12 hours and take pictures,ImageJ software were utilized to calculate the scratch area;8 Invasion experiment:Transwell cell method was used,Matrigel glue was evenly spread on the bottom of the cell layer,and A549 cells and H1299 cell suspension of transfected control/HMG2L1-shRNA lentivirus were inoculated into the cell,using 1%FBS complete medium for starvation up to 24h,replace the outside medium with 10%FBS complete medium,incubate at37°C for 24 hours,take out the chamber,fix and stain the cells outside the bottom of the chamber,and take photos to calculate the number of cells;9 Research on cell signaling pathway:inoculate A549 cells and H1299 cells transfected with control/HMG2L1-shRNA lentivirus into 6-well plates,collect cells to extract total protein when the cells grow to 80%fusion,and use Western blot to detect cell signals Pathway related protein expression.10.Statistical analysis:prism 6.0 software is used for statistical analysis of data.Numerical variables are expressed as mean±standard deviation.Comparison between the two groups should use independent sample t test.Comparison between multiple groups should use one-way analysis of variance(one-way analysis of variance,ANOVA).Categorical variables are expressed as percentages,and chi-square test(chi-square test)is used for comparison between groups.test.P<0.05 indicates statistical difference.Results:Expression and clinical significance of HMG2L1 in NSCLC:1.Expression of HMG2L1 in lung cancer tissues and adjacent tissues of patients with NSCLC:A total of 60 pairs of cancer tissue and adjacent para-cancerous tissues were collected and tested.The HMG2L1 detection level was as follows:H-SCORE of cancer tissue was 176±4.71,H-SCORE of adjacent para-cancerous tissue was 84.68±4.713,the difference was statistically significant(P<0.0001).2.The relationship between the expression level of HMG2L1 and clinicopathological characteristics:Taking the median H-score score(179.8)of cancer tissue samples as the boundary,the expression of HMG2L1 in Lung cancer tissues was divided into low expression group(≦179.8)and high expression group(>179.8),the results showed that the expression level of HMG2L1 was not significantly correlated with the patient’s gender,age,smoking status,tumor size,pathological type,lymph node metastasis,but was related to the degree of differentiation of the pathological tissue and clinical stage,indicating The higher the expression level of HMG2L1,the lower the degree of differentiation of the pathological tissue and the later the clinical stage;Using the t test to further analyze the expression level of HMG2L1 in each clinical feature group,it was found that the expression level of HMG2L1 may be related to gender,smoking,pathology types.3.Until March 30,2020,the average follow-up time was 16.2 months of the 60 patients,(7-21ms).Of the 60 patients,38 patients did not receive chemo-radiotherapy and other tumor-related treatments(38/60,63%),and 22 patients(22/60,37%)received 1-8 cycles of chemotherapy.There were 12 cases of disease progression(12/60,20%),including 4 deaths(4/60,6.7%).The H-score score of 12 patients with disease progression was 190±5.359,2cases in stage IA(2/60,3.3%),2 cases in stage IB(2/60,3.3%),and 3 cases in stage IIA(3/60,5%),3 cases of stage IIIA(3/60,5%),2 cases of stage IIIB(2/60,3.3%);4 cases did not undergo postoperative chemotherapy(4/60,6.7%),8 cases performed Postoperative chemotherapy(8/60,13.3%).The average H-score score of 4 dead patients was 200.6±5.086,1case in stage IIA(1/60,1.7%),2 cases in stage IIIA(2/60,3.3%),and 1 case in stage IIIB(1/60,1.7%),the patient of stage IIA didn’t receive chemotherapy,the other 3 patients all received chemotherapy.So far,the high expression of HMG2L1 may be associated with poor survival prognosis(P<0.05),and no significant difference in Disease-free survival(DFS)(P>0.05).The function and mechanism of HMG2L1 in NSCLC:1.Among the three primer sequences,HMG2L1-shRNA-VL1222 can significantly knock down HMG2L1,the lentivirus were transfected into A549 cells and H1299 cells,and then successfully obtained the HMG2L1 interfered cell line through BSD screening.Western Blot and qRT-PCR Verifed that the expression of HMG2L1 gene and protein is significantly down-regulated after transfection;2.The effect of HMG2L1 on the proliferation of NSCLC cells:the cell proliferation rate decreased after knocking down HMG2L1 in A549 cells,and had a significant difference on the6th day of culture(P<0.001);There was also a decrease in cell proliferation rate of H1299-HMG2L1-shRNA,and there was a significant difference on the 6th day of culture(P<0.001).Comparing the proliferation rate of the two cell lines HMG2L1,the A549 cell seems to decrease significantly.3.The effect of HMG2L1 on the migration ability of NSCLC cells:The results of the cell scratch test showed that A549 cells migrated slowly,and it took nearly 68-90h for the scratches to fully close;H1299 migrated faster,around 36 hours of move to the opposite side.The migration rate of A549 cells and H1299 cells transfected with HMG2L1-shRNA was slower than that of the control group,and the difference was statistically significant(P<0.01).4.The effect of HMG2L1 on the invasion ability of NSCLC cells:Transwell chamber experiment showed that knocking down HMG2L1 in A549 cells can significantly reduce the cell invasion ability(P<0.01),while in H1299,the transfection of HMG2L1-shRNA The ability of H1299 cells to penetrate Matrigel significantly decreased(P<0.01).5.Explore the mechanism of HMG2L1 in NSCLC cells:after knocking down HMG2L1 in A549 cells and H1299 cells,althoughβ-catenin and Wnt5a have a slight upward trend,but there is no statistical significance(P>0.05)There was no obvious change in STAT3 expression,which may suggest that the function of human HMG2L1 in NSCLC cells does not play a role through Wnt signaling pathway and STAT3.Therefore,the mechanism of HMG2L1 in NSCLC needs further exploration.Conclusions:1.In patients with NSCLC,the expression of HMG2L1 is significantly higher in lung cancer tissues than in adjacent para-cancerous tissues;2.The expression level of HMG2L1 is related to the degree of NSCLC differentiation and clinical stage of pathological tissue.The higher the expression level of HMG2L1,the lower the degree of pathological differentiation and the later the clinical stage;at the same time,it may also be related to gender,smoking,pathology.The expression levels of males,smoking and squamous cell carcinoma would be higher.3.The expression level of HMG2L1 may be related to the survival prognosis of NSCLC patients.The higher the expression of HMG2L1,the shorter the overall survival period.So far,no correlation between HMG2L1 expression level and progression-free survival has been found.4.HMG2L1 can promote the proliferation,migration and invasion of NSCLC,and may be involved in the occurrence and metastasis of lung cancer.5.The mechanism of HMG2L1 in NSCLC may not be related to Wnt signaling pathway moleculesβ-catenin,Wnt5a and STAT3,and may play a role through other signaling pathways. |
