The Expression Level Of LIN28B And Its Relationship To Long-term Outcomes For Gastric Cancer Patients

Background: Gastric cancer(GC)is referred as one of the most common digestive system malignancies around the world,causing 697,000 new diagnosis and 499,000 related deaths each year in China [1].The incidence rate and mortality of GC in our country are more than two times beyond the average rates of other countries,which means the absolute number of newly diagnosed patients is the highest worldwide[2].Recently,the combination of surgery and adjuvant chemotherapy is the routine strategy for gastric cancer management,and so far,the radical surgery is still the only cure method.Most patients(80-90%),however,have developed advanced gastric cancer(AGC)at the first consultation due to the sufficient of medical conditions and the lack awareness of general survey in our country,suffering from the high post-operation recurrent rate and metastasis rate and even the failure to receive radical surgery [3,4].The five-year survival rate remains unsatisfactory(<40%)for gastric cancer patients who received radical surgery and/or effective radio-chemotherapy[5].Based on the overmentioned,the further exploration of the biochemical characteristics of gastric cancer and the discovery of some genes related to metastasis,recurrence,progress,and drug-resistance is of great significance for further improving the comprehensive curativeeffectiveness and short-/long-term prognosis of gastric cancer.Recent studies have shown that LIN28 B is a gene related to the development and progress of malignant diseases,located on chromosome 6q21 in the human genome and having a coding region contains 250 amino acid residues,which is highly similar to the LIN28 A sequence(76%)[6].The protein encoded by LIN28 B gene is a highly conserved RNA-binding protein that possesses the ability to recode human fibroblasts into pluripotent stem cells and plays an extremely important role in regulating cell growth,differentiation,metabolism and pluripotency[7].Many articles have revealed that LIN28 B protein is highly expressed in stem cells and embryonic,meanwhile,with the continuous differentiation and maturation of them,the expression level of LIN28 B protein is reduced gradually.In some human malignant tumors,however,the abnormally high expression of LIN28 B protein has been witnessed,playing an essential role in the maintenance of stem-like cells[8,9].Now researchers have suggested that LIN28 B performs its biological functions mainly by regulating let-7 micro RNA through the double-negative feedback loop [10].As a hot spot in research,mi RNAs are small non-coding RNAs that bind the m RNA of target genes to inhibit their translation and/or induce their decay.mi RNAs thus play a crucial role in many biological events after binging the target m RNAs,including cell differentiation,proliferation,apoptosis,metabolism,and stress.Recent research not only revealed that the dysregulation of mi RNAs plays an important role in tumorigenesis but also highlighted that its low-expression often occurs in malignant tumors[10,11].LIN28 B negatively regulates lei-7 family mi RNAs via inhibiting their post-transcriptional maturation to achieve its cancer-promoting function.The let-7 family of mi RNAs,identified in the human genome in 1993,is the largest of all mi RNA families and the remarkable factor in the maintaining of embryonic stem cell differentiation and plasticity[10,12].On the other hand,accumulating evidence has suggested that LIN28 B is highly related to the formation of tumor stem cell and an important molecular marker on its surface[13].Studies published recently not only revealed that LIN28 B recodes fibroblasts into pluripotent stem cells but also showed that it maintains the stability of stem-like cells in tumors.Therefore,it's very likely that LIN28 B is involved in the occurrence and development of gastric cancer.Metastasis and recurrence are realized as the main reason for cancer-related deaths in patients with gastric cancer.The up-regulation of LIN28 B is associated with recurrence of gastric cancer.Chemotherapy is an indispensable part of comprehensive treatment for gastric cancer,with oxaliplatin as the first-line drug.But the drug-resistance to oxaliplatin in gastric cancer is very common and seriously weakens the effectiveness of chemotherapy.Thus,it's likely that finding the underlying mechanism of drug-resistance may reduce metastasis and recurrence in gastric cancer,which allows the patients to have a better longterm outcome.Part One: The relationship between LIN28 B expression level in gastric cancer tissues and the clinicopathological characteristics and prognosis in gastric cancer patients Purpose: This part was designed to evaluate the correlation between LIN28 B expression level in gastric cancer tissues and the characteristic of pathology,TNM classification,differentiation and long-term outcomes in gastric cancer patients.Methods: 1)From 2005 to 2012,the entire clinical records and tumor tissues from 250 patients with gastric cancer were retrospected(100 cases from Jinling Hospital and 150 from Xijing Hospital);2)HE staining and immunohistochemistry;3)Semi-quantitative analysis and classification of immunohistochemical staining;4)?2 and t test for the relationship between LIN28 B expression and patients' characteristic,Kaplan-Meier for survival curve.Results: 1)LIN28B protein expression was low in 96 patients(38.4%)and high in 154 cases(61.6%);2)The relationship between LIN28 B protein expression and differentiation scales was significant(P=0.001),which suggested that the higher expression indicates poorer differentiation;3)The 5-year survival rate in the low expression patients was better than that in the high expression patients(P<0.01);4)Multivariate analysis showed that the LIN28 B protein expression level and N classification(lymph node metastasis)are independent predictor factors.Conclusion: The expression level of LIN28 B protein in gastric cancer tissues is related to tumor differentiation rather than tumor size and invasion.The high expression level usually means poor outcomes.The LIN28 B protein expression level and N classification are independent predictor factors.Part Two: The expression of LIN28 B in paired clinical samples of gastric cancer and adjacent normal counterpart,and the relationship between its expression in cancer tissues and the pathological response after neoadjuvant chemotherapyPurpose: This part was designed to evaluate the difference in the LIN28 B expression between cancer tissues and the matched normal tissues.And then the effects of LIN28 B expression level in tumor tissues on neoadjuvant chemotherapy and the change of LIN28 B expression level before and after chemotherapy were analyzed.Methods: 1)From 2015 to 2017,twenty-four new diagnosed gastric cancer patients were enrolled at Jinling Hospital;2)Before NAC,abdominal enhanced CT scan was used for clinical evaluation,and gastric cancer tissues and normal tissues were collected by gastroscopy;3)All patients received D2 radical surgery before enhanced CT review following 2 cycles of neoadjuvant chemotherapy;4)Tumor response to chemotherapy was evaluated according to Response Evaluation Criteria in Solid Tumors(RECIST)guideline [14],meanwhile,pathological response was analyzed according to Becker criteria [15];5)The expression level of LIN28 B RNA and protein were evaluated by RT-PCR and immunohistochemistry;6)The relationship between LIN28 B expression and clinical/pathological response to chemotherapy was assessed by Fisher test,meanwhile,the difference in the LIN28 B expression between cancer tissues and the matched normal tissues was discovered by paired t test.The change of LIN28 B expression before and after chemotherapy was also evaluated.Results: 1)There was no relationship between LIN28 B expression and clinical response to chemotherapy(P=0.432),with 11 cases in the low-expression group(PR 5,SD 5,and PD 1)and 13 in the high-expression group(PR 7,SD 3,and PD 3);2)The relationship between LIN28 B expression and pathological response to chemotherapy was found(P=0.006),with 11 cases in the low-expression group(5 cases in grade 1a,8 in grade 1b-2,and 1 in grade 3)and 13 in the high-expression group(0 case in grade 1a,5 in grade 1b-2,and 8 in grade 3);3)Before neoadjuvant chemotherapy,there were 9 cases in the low-expression group and 13 cases in the high-expression group.But after neoadjuvant chemotherapy,the number of patients was increased up to 17 cases in the high-expression group and decreased to 5 cases in the low-expression group.The change was statistically significant(P=0.002);4)The expression level of LIN28 B in gastric cancer tissues was higher than that in matched normal tissues(3.87 ± 0.35 vs.1.95 ± 0.20,P<0.01).Conclusion: The expression level of LIN28 B in gastric cancer tissues was higher than that in matched normal tissues,which was correlated with pathological response to neoadjuvant chemotherapy rather than clinical response.It means that gastric cancer patients with higher LIN28 B expression level may suffer from the poor pathological efficacy of NAC.Thus,the LIN28 B expression can be a predictor factor for the efficacy of NAC.The reason for the up-regulation of LIN28 B after NAC is that instead of other positive cells,tumor cells with negative LIN28 B expression is more likely to be sensitive to chemotherapy and killed by the treatment which means the increased ratio of positive cells.Part Three: Construction stable expression LIN28 B protein gastric cancer cell line and evaluate the relationship between LIN28 B expression and proliferation,invasion and chemoresistance in vitroPurpose: This part was designed to establish stable expression LIN28 B gastric cancer cell clone and evaluate the relationship between its expression and proliferation,invasion and chemoresistance.Methods: 1)Three gastric cancer cell lines(SGC7901,SGC823 and MKN45)were selected and examined the expression of LIN28 B RNA and protein via RT-PCR and Western blot.The negative expression cell line(SGC7901)was then selected for the further experiment.2)The plasmids pc DNA3.1+-LIN28 B and pc DNA3.1+ were transfected into SGC7901 cells,and then the expression of LIN28 B was identified by RT-PCR and Western blot;3)The proliferation,invasion,and chemoresistance were tested by MTT,cell clony formation,transwell assays and cell viability analysis;4)Knockdown the LIN28 B expression level of high-expression cells clone by si RNA interference technology,the influence of down-regulated LIN28 B was evaluated by MTT,cell clony formation,transwell assays and cell viability analysis;5)ANOVA and t test were used to analyze the difference between groups.Results: 1)After stable transfection,the expression of LIN28 B RNA and protein in SGC7901-LIN28 B clone was dramatically increased than the control group(P<0.01);2)The SGC7901-LIN28 B clone had more ability of proliferation(P<0.05),invasion(P<0.01),and chemoresistance(P<0.05)than the control group;3)After si RNA interference,the expression of LIN28 B was decreased in highexpression cells clone and the ability of proliferation,invasion,and chemoresistance was suppressed.Conclusion: After stable transfection,the expression of LIN28 B RNA and protein in gastric cancer cells was increased,meanwhile the cell viability,proliferation,and drug-resistance were improved,which makes sense for that the up-regulation of LIN28 B promotes cancer cells proliferation,invasion and chemoresistance to affect the long-term outcome of patients with gastric cancer.