Effect Of Long No Coding MALAT1 On Pancreatic Ductal Adenocarcinoma’s Biological Function And Its Mechanism

Pancreatic cancer(PC) is one of the malignant tumors attracting more concern in the world. Because of its higher malignant degree, early metastasis, less chance of operation, easy recurrence and high mortality rate, PC is known as "the king of cancer". In 2015, the mortality rate of PC was the ninth in cancer deaths reasons in China. According to the forecast of US National Cancer Center, the death rate will be increased to the second place in the cancer death ranking list until 2030. Pancreatic ductal adenocarcinoma(PDAC) accounts for over 90% and the post-operation median survival time of PDAC patients are less than six months. In spite of the improvement of diagnosis and treatment means, no significant improvement is found in the prognosis of PDAC patients, and the five-year survival rate is only about 7%. Therefore, the importance of our indepth study of PDAC is self-evident.In recent years, it has been demonstrated that long non-coding RNAs(Lnc RNAs) are involved in the pathophysiology of human normal and abnormal developmental processes; and closely linked to many human diseases including cancer, and control the occurrence, development and prognosis of cancer in several ways. Metastasis Associated Lung Adenocarcinoma Transcript1(MALAT1) is one of the earliest discovered Lnc RNAs, which located on chromosome 11q13.1 and its transcript length is 8708 bp.Compared with the other normal tissues, the expressions of MALAT1 were relatively high in pancreas, lung tissues. In NSCLC, pancreatic cancer, prostate cancer tissues, MALAT1 was over-expressed compared with their precancerous lesions. Its expression level was negatively related to the overall survival time. MALAT1 played the role in promoting cell proliferation, migration and invasion in the development of cancer. However, rare study had been reported concerning the expression of MALAT1 in PDAC, and the mechanism unclear. Therefore, our study will focus on the relationships between the MALAT1 expression and clinicopathological parameters, and the influence of MALAT1 on PDAC cell’s biological functions and its molecular mechanism.The level of MALAT1 m RNA was detected in forty-five PDAC formalin fixed paraffin embedded(FFPE) tissues and twenty-five FFPE adjacent normal tissues(ANT) by q RT-PCR. The result showed that the expression level of MALAT1 m RNA in PDAC tissues was significantly higher than that in ANT tissues(P=0.009). The positive rate of MALAT1(77.8%, 35/45) in PDAC tissues was significantly higher than that(32%, 8/25) in ANT by using in situ hybridization(ISH)(P=0.0019). MALAT1 m RNA level was also tested in PDAC cells(PANC-1, MIAPACA-2, BXPC-3, CANPAN-1, ASPC-1) and immortalized cell(HPDE6C-7). MALAT1 m RNA level in ASPC-1 was significantly higher than that in HPDE6C-7(P<0.05), while the MALAT1 m RNA level in BXPC-3, MIAPACA-2 and CANPAN-1 significantly lower than that in HPDE6C-7(All P<0.05). However, there was no significant difference of MALAT1 level between PANC-1 and HPDE6C-7(P>0.05).In the analysis of clinicopathological parameters, the expression of MALAT1 m RNA was compared in groups according to tumor size(<4cm, ≥4cm), invasion depth(T1, T2 and T3, T4) and clinical stage(I, II and III, IV) in 45 PDAC tissues. The expression of MALAT1 was significantly higher in≥4cm group than that in the <4cmsize group(P=0.036), the expression of MALAT1 in the deep invasion group(T3/T4) was significantly higher than shallow invasion group(T1/T2)( P=0.014), and the MALAT1 level in the advanced clinical stage(Stage III and IV) was significantly higher than that in the early clinical stage(Stage I and II)(P=0.0328). Furthermore, according to 45 PDAC cases were divided into high-expression group and low-expression group by the average value of MALAT1 in q RT-PCR, the relevant analysis of the related clinical parameters showed that the expression of MALAT1 was positively related to tumor size, invasion depth and oncology clinical stage(r=0.35,P=0.019;r=0.334,P=0.025;r=0.439,P=0.04). While there were no significantly correlated with the clinical factors such as patient sex, age, tumor location, tissue type classification, lymph node metastasis, vein invasion, nerve invasion, distant metastasis, tumor serum CEA level and tumor serum CA199(All P>0.05). The survival curve drawn by the Kaplan-Meier method showed that patients with high expression of MALAT1 had shorter survival time than that of the patients with low expression of MALAT1( P<0.043). The analysis of univariate analysis by log-rank test showed that PDAC lesion location(HR=2.103, 95%CI: 0.98-4.650, P=0.013), invasion depth(HR=2.136, 95%CI: 0.999-4.965, P=0.021) and expression of MALAT1 m RNA(HR=1.505, 95%CI: 1.027-2.205, P=0.036) were independent prognostic factors. By incorporating the clinical factors with univariate result P≤ 0.01 into the Cox regression model, the analysis showed that lesion location of PDAC(HR=3.482, 95%CI: 1.414-8.547, P=0.007), expression o f MALAT1 m RNA(HR=1.798, 95%CI: 1.177-7.747, P=0.007) and nerve invasion(HR=4.631, 95%CI: 1.86-11.553, P=0.001) were independent prognostic factors. In addition, the ROC curve showed that the AUC was 0.69(95% CI:0.561-0.829, P=0.009), the cutoff value was 0.1035.The diagnostic sensitivity and specificity were 77.8% and 60%, respectively.In vitro, we designed MALAT1-sh RNA lentiviral vector. By means of positive cloning, PCR identification and sequencing analysis, the result showed that 4 pairs of sh RNA chains respectively aiming at MALAT1 have connected with plasmid p GLV3/H1/GFP+Puro and constructed the lentivirus MALAT1-sh RNA expression vector successfully. After package, purification, and concentration, the generated lentivirus titer was 1×108TU/m L. 4 pairs of MALAT1-sh RNAs had been transfected with PANC-1 and MIAPACA-2 cells, with the inhibition rate of respectively 14%~61% and 17.5%~73.6%, among which, MALAT1-5416 and MALAT1-5543 had the most significant declination, with 61% and 73.6%, respectively. By the puromycin screening, we had successfully constructed the PANC-1 and MIAPACA-2MALAT1 knockdown stable cell lines; the knockdown rate of MALAT1 in PANC-1 and MIAPACA-2 are respectively 63% and 72% by q RT-PCR.The result of CCK-8 showed that the proliferation of PANC-1 and MIAPACA-2 group(knocking down MALAT1) was significantly slower than that of blank control group and negative control group(P<0.001). The flatbed cloning experiment showed that the cloning and formation ability of PANC-1 and MIAPACA-2 group(knocking down MALAT1) was significantly weaker than that of blank control group and negative control group(P<0.05). The migration experiment by Transwell showed that the number of PANC-1 and MIAPACA-2 cells(knocking down MALAT1) migrating to the lower surface of membrane was significantly less than that of blank control group and negative control group(P<0.05);while the invasion experiment by Transwell cell showed that the cell-penetrating number of PANC-1 and MIAPACA-2 after the knockout of MALAT1 was significantly less than that of blank control group and negative control group(P<0.05). By the flow cytometry to test apoptosis, the experiment result showed that the early apoptosis rate of PANC-1 and MIAPACA-2 was respectively 26.93%3±1.74% and 58.7%±4.4%, and significantly higher than that of the blank control group or negative control group(P<0.05). By using the flow cytometry to test the cell cycle, there was no significant difference in the proportion of DNA betweent PANC-1and MIAPACA-2 group after the knockdown MALAT1 compared with blank control group or negative control group(P>0.05). In vivo, knockdown of MALAT1 will inhibit the growth of PANC-1 cell’s proliferation. After knockdown MALAT1, the absorbance of the transcription factor FOS protein MIAPACA-2 cell by using of WB, dropped by 49.4%. The FGF2 protein expression significantly increased and the absorbance value of CAV1 protein increased by 310.1%, while the absorbance of FOS protein in the PANC-1 cell dropped by 30.6%, the absorbance of the FGF2 protein increased by 67% and the absorbance of CAV1 protein increased by 74%, while there was no significant change in other proteins such as EGFR, ITGA6, VECFC, MET and RAP1, which suggested that MALAT1 can regulate PDAC cell’s biological functions such as growth, invasion, metastasis and apoptosis by the regulating FOS/FGFR2 or CAV1 gene expression.In this experiment, the expression of MALAT1 increased in the later stage by lentivirus MALAT1-sh RNA knockdown and knockdown rate of MALAT1 was unstable. Therefore, this study has been applied CRISPR/Cas9 to knock out and activate MALAT1 in diploid HCT116 WT cells of the colorectal cancer(CRC). This technology can overcome the defects of the other transfection methods(si RNA/sh RNA) with lower disturbance efficiency, the increase of target gene in the later stage, Off-Target effect and shortcoming of TALEN activation system. In this study, it was the first time to construct MALAT1 knockout system by using dual-g RNA and activate over-expression system of MALAT1 by using d Cas9-sg RNA in HCT116 WT cell. In this experiment, we had been successfully constructed the MALAT1 gene-specific CRISPR/Cas9 vector and stable cell lines of knock out and activated MALAT1. The efficiency of knock out MALAT1 by dual-g RNA was up to 99% and the efficiency of activated MALAT1 by d Cas9-sg RNA was increasing 5-7 times in HCT116 WT cell. In vitro, after knocking out and activating MALAT1 by CRISPR/Cas9, The ability of proliferation, cloning in HCT116 WT cell was respectively inhibited and enhanced compared with the control group(P<0.05). After knocking out and activating MALAT1, the expression of twist and snail m RNA was respectively dropped and increased compared with the control group(P<0.05).After MALAT1 was rescued in HCT116 WT knockout MALAT 1 cell, the m RNA levels of twist and snail were higher than that of the control group(P<0.05).The protein level of P65 in knockout MALAT group did not change, while the protein level of P65 in control group was increased via TNFa induced, which suggested that the growth, proliferation and migration of HCT116 WT cell can be inhibited by inactivating NF-k B channel and decreasing nucleus of twsit and snail.To sum up, the high expression of MALAT1 was in tissue and some cells of PDAC. The expression of MALAT1 m RNA was positive correlated with tumor size, invasion depth and clinical stage. The expression of MALAT1 was negatively related to survival time. MALAT1 can be regarded as diagnosis indictor and independent prognostic factor. In vitro, the proliferation, invasion and migration of PDAC cells were inhibited and induced early apoptosis after knocking out MALAT1. In vivo, the proliferation and tumorigenic ability of PANC-1cell was inhibited after knock down MALAT1. Knock down MALAT1 in PDAC cells, protein level of FOS was deceased, and protein level of CAV1 and FGR was increased which indicated that MALAT1 regulate and control proliferation, invasion and migration of PDAC cell through FOS-FGFR2/FGR or CAV1 axis to activate ERK/MAPK signal pathway. At the same time, we have been successfully constructed knock out MALAT1 system by dual-g RNA and MALAT1 over-expression system by activating d Cas9-sg RNA. CRISPR/Cas9 technology will bring new wishes for PDAC gene study and targeted therapy.