Adoptive Transfer of NKG2D CAR mRNA-Engineered Natural Killer Cells in Colorectal Cancer Patients.

By fusing the extracellular domain of the natural killer (NK) cell receptor NKG2D to DAP12, we constructed a chimeric antigen receptor (CAR) to improve NK cell tumor responses. An RNA electroporation approach that provides transient expression of the CAR was adopted as a risk mitigation strategy. Expression of the NKG2D RNA CAR significantly augmented the cytolytic activity of NK cells against several solid tumor cell lines in vitro and provided a clear therapeutic benefit to mice with established solid tumors. Three patients with metastatic colorectal cancer were then treated with local infusion of the CAR-NK cells. Reduction of ascites generation and a marked decrease in number of tumor cells in ascites samples were observed in the first two patients treated with intraperitoneal infusion of low doses of the CAR-NK cells. The third patient with metastatic tumor sites in the liver was treated with ultrasound-guided percutaneous injection, followed by intraperitoneal infusion of the CAR-NK cells. Rapid tumor regression in the liver region was observed with Doppler ultrasound imaging and complete metabolic response in the treated liver lesions was confirmed by positron emission tomography (PET)- computed tomographic (CT) scanning. Our results highlight a promising therapeutic potential of using RNA CAR-modified NK cells to treat metastatic colorectal cancer.

miR-145 suppresses colorectal cancer cell migration and invasion by targeting an ETS-related gene.

MicroRNA-45 (miR-145) has been demonstrated to be downregulated in various cancer types including colorectal cancer (CRC). However, the function of miR­145 in CRC has not been clearly elucidated. In this study, we examined miR-145 expression by quantitative real­time PCR (qRT­PCR) in CRC cell lines as well as tumors and corresponding normal mucosa, and the results were correlated to the clinicopathological parameters. In addition, using computational algorithms we investigated putative miR­145 targets. The role of miR­145 was further examined in studies in vitro. In our study miR­145 was significantly decreased in CRC tissues and cell lines compared with non­cancerous colorectal mucosa, especially lymph node or distance metastasis cases. Based on computational algorithms, we assumed that ERG was directly modulated by miR­145 in colorectal cancer cells. For the first time, we demonstrated that ERG was highly expressed in CRC tissues compared with normal ones by qRT­PCR. The inverse correlation between the expression of miR­145 and ERG was observed in CRC tissues. Dual­Luciferase assays demonstrated the direct interaction between miR­145 and 3'­UTR of ERG mRNA. Ectopic expression of miR­145 suppressed the proliferation and invasion ability of colorectal cancer cells, while ERG knockdown partially restored the tumor suppressive effect of miR­145. These results suggested that miR­145 might act as a tumor suppressor during the process of CRC malignant transformation by interacting with ERG.

MicroRNA-490-3p inhibits colorectal cancer metastasis by targeting TGFßR1.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignances worldwide. Metastasis is responsible for the rapid recurrence and poor prognosis of CRC. However, the underlying molecular mechanism of CRC metastasis remains largely unclear. In this study we purposed to investigate the expression and biological functions of miR-490-3p in CRC metastasis, as well as to identify its downstream target genes and influenced pathway. METHODS: The expression level of miR-490-3p in CRC cell lines, CRC adjacent normal tissues, non-metastasis and metastasis tissues were assessed by quantitative real-time PCR. Patient survivals were follow-up up to 7 years. Gain-of-function and loss-of-function study on cell migration and invasion abilities were carried out by transfection of miR-490-3p mimics or inhibitors respectively. The molecular targets of miR-490-3p were computationally identified and experimentally verified by dual-luciferase reporter assay and western blot. Functional rescue was also conducted to confirm miR-490-3p inhibits CRC metastasis by targeting TGF-ß signaling pathway. RESULTS: miR-490-3p expression was persistently downregulated during CRC malignant progression, as well as in CRC cell lines. Artificially overexpression miR-490-3p in CRC cell lines inhibited cell migration and invasion abilities while knockdown miR-490-3p expression caused the reverse effects. TGFßR1 and MMP2/9 were the downstream targets of miR-490-3p in CRC. Inhibition of TGFßR1 could partially recover the tumor suppression effect of miR-490-3p. CONCLUSION: miR-490-3p is downregulated during CRC malignant progression. miR-490-3p represses CRC cell migration and invasion abilities, partially by targeting to the TGF-ß signaling pathway. Taken together, miR-490-3p is acting as a tumor suppressor in CRC.

Downregulation of microRNA-1 and microRNA-145 contributes synergistically to the development of colon cancer.

The aim of the present study was to identify the differentially expressed microRNAs (miRNAs or miRs) in patients with colon cancer and examine their roles in the pathogenesis of the disease. The expression profiles of miRNAs were examined in tumor tissue samples collected from 20 patients with histologically confirmed colon cancer and in the adjacent non-cancerous control tissues using microarray analysis. We found that numerous miRNAs were differentially expressed in the colon cancer tissues compared with the control tissues. Following functional analysis, we noted that three miRNAs which were significantly downregulated, miR-145, miR-451 and miR-1, shared the same target gene, [NLR family, apoptosis inhibitory protein (NAIP)], which has previously been reported to be involved in the development of colon cancer. We then confirmed that NAIP is a target gene of miR-145 and miR-1, but not of miR-451, using a luceriferase assay, and we confirmed the expression patterns of these miRNAs and NAIP in the tumor tissue, as well as in the adjacent non-cancerous control tissues. Additionally, in order to examine the function of miR-145 and miR-1 in human colon cancer, we transiently transfected a human colon cancer cell line with miR-145 and miR-1 mimics or inhibitors, and the results of MTT assay revealed that the re-expression of miR-145 and miR-1 inhibited the survival of colon cancer cells, which may be attributed to the inhibition of the anti-apoptotic activity of NAIP. Our findings demonstrated that miR-145 and miR-1 play a negative regulatory role in the proliferation of colon cancer by targeting NAIP; thus, miR-145 and miR-1 may prove to be novel therapeutic targets in the treatment of colon cancer.

The potential value of miR-1 and miR-374b as biomarkers for colorectal cancer.

The mortality of colorectal cancer (CRC) is growing due to the unsatisfactory specificity and sensitivity of the existing screening methods. Previous studies have focused on the role of miRNAs as CRC biomarkers. However, few studies have examined the miRNA profiles at each stage. The objective of this study was to identify miRNAs that distinguish CRC patients from normal people to prevent the misdiagnosis of patients with certain stages of CRC. We performed miRNA profiling of 1547 human miRNAs by qRT-PCR in CRC patients with stage II and stage III disease. The statistical analyses showed that there were 96 miRNAs that were significantly dysregulated in CRC relative to normal tissues (P<0.05). There were 28 dysregulated miRNAs associated with separate or combined stages II and III disease. There were 25 downregulated miRNAs, including the following: miR-1, -145, -145*, -137, -363, -143, -4770, -490-5p, -9, -144*, -99a, -99b, -23b, -143*, -100, -768-3p, -24-1*, -125a-5p, -30e*, -574-3p, -126, let-7b, miR-1979, -374b, and -140-3p. We found an upregulation of miR-203, 182, and 96. Our results demonstrated that the expression of miR-1 and miR-374b was significantly decreased in each stage and may function as a biomarker of CRC. Furthermore, 20 miRNAs were dysregulated both in stage II disease without lymph node or distant metastasis and in stage II-III tumors but not in stage III tumors. Only miR-4794 was involved exclusively with stage II tumors, and there were 19 miRNAs that were dysregulated only in stage III disease with lymph node metastasis and in stage II-III disease. There were only 6 miRNAs that were uniquely dysregulated in stage III. Our results indicate that miRNA expression may be valuable in the clinic. However, large prospective studies are required to confirm the role of miRNAs. This study provides a new model for analyzing novel CRC biomarkers by considering more clinical factors.

Identification and validation of potential biomarkers for the detection of dysregulated microRNA by qPCR in patients with colorectal adenocarcinoma.

Colorectal cancer represents a lethal disease that has raised concern and has attracted significant attention. Adenocarcinoma is the most common type of colorectal cancer (CRC). MicroRNAs are thought to be potential biomarkers of CRC. Many researchers have focused on the expression pattern of miRNAs in CRC. However, previous studies did not pay particular attention to the effects of the degree of differentiation of the cancer with respect to the miRNA expression profile. First, this study compared the expression level of 1547 miRNAs by qRT-PCR in Colorectal adenocarcinoma tissues to that in paired normal tissues. In all, 93 miRNAs were identified that were significantly dysregulated in Colorectal adenocarcinoma relative to normal tissues (P<0.05). Then, we analyzed their potential as cancer biomarkers by ROC analysis, and the result revealed that three miRNAs with high sensitivity and specificity are suitable as biomarkers for the diagnosis of CRC (the value of the AUC was greater than 0.7). Interestingly, previous reports of 23 of these miRNAs have been scarce. Furthermore, we wanted to analyze the difference between well- and moderately differentiated cancers, and as expected, 58 miRNAs showed significant dysregulation. Importantly, 32 miRNAs were able to not only distinguish cancer tissues from normal tissues, but they were also able to identify well- and moderately differentiated cancers. In conclusion, the degree of differentiation has an important influence on the miRNA expression pattern. To avoid misdiagnoses and missed diagnoses, tumors of different degrees of differentiation should be treated differently when miRNAs are used as cancer biomarkers.

Identification of miRNAs differentially expressed in clinical stages of human colorectal carcinoma-an investigation in Guangzhou, China.

Aberrant expression of microRNAs (miRNAs) has been implicated in human cancer, including colorectal cancer (CRC). Such dysregulated miRNAs may have potential as diagnostic markers or therapeutic targets. However, the nature of an association between these miRNAs and clinical stages of CRC is still not clear. To this end, we performed a miRNA profiling of 1547 distinct human miRNAs using 31 samples of tumor and paired normal mucosa obtained from 31 CRC patients. Based on statistical analyses of profiling data, we identified 569 miRNAs that were significantly dysregulated in CRC relative to normal tissues (P<0.05). Among the 569 dysregulated miRNAs, downregulation of 17 was associated with stages II, III, and IV colon and rectal cancers (separate or combined), according to our criteria. We also assessed the potential of these dysregulated miRNAs as diagnostic biomarkers for CRC patients who were without metastasis, and the value of the dysregulated miRNAs for predicting metastasis, lymph node and distant. Their distinct expression patterns in colon and rectal cancers were also examined. Although our findings cannot be immediately applied toward clinical diagnosis, our new study model for determining and assessing the biomarker potential of dysregulated miRNAs should be useful in further research in detection of human CRC.

[Study on miR-490-5p and miR-363 as novel biomarkers for the diagnosis of colorectal cancer].

OBJECTIVE: To identify dysregulated microRNA(miRNA) that can act as novel biomarker for colorectal cancer screening. METHODS: Real time-polymerase chain reaction was used to detect the expression profile of miRNA in tumor and paired normal tissues, and the significant dysregulated miRNA was examined by non-paired t-test. Receiver operating characteristic(ROC) curve was performed to evaluate the specificity and sensitivity of miR-363 and miR-490-5p as biomarkers to discriminate colorectal cancer patients from normal person. RESULTS: Seven-three dysregulated miRNAs were found in male samples, of which 6 miRNAs were up-regulated and other 67 miRNAs were down-regulated, while 42 dysregulated miRNAs were found in female samples, of which 5 were up-regulated and 37 were down-regulated in tumor tissues compared with normal tissues. Among above dysregulated miRNAs, 33 miRNAs had significant differences, besides, dysregulated expression level of 10 of 33 miRNAs was over 5 folds in male and female as well as mixed samples. CONCLUSIONS: The expression pattern in female is different from that in male. miR-363 and miR-490-5p possess the potential in screening colorectal cancer patients from healthy people.

[Role of centromere protein H in human gastric cancer cell proliferation].

OBJECTIVE: To explore the role of centromere protein H (CENP-H) in the proliferation of human gastric cancer cells. METHODS: RT-PCR and Western blot analysis were employed to examine the mRNA and protein expressions of CENP-H in 7 human gastric cancer cell lines and immortalized human gastric epithelial cells (GES-1). The cells were infected with the retrovirus vectors pMSCV-CENP-H or CENP-H-RNAi to establish stable cell lines with high CENP-H expression or CENP-H expression interference. MTT assay and colony formation assay were used to examine the changes in the cell proliferation after the infection. RESULTS: CENP-H was over-expressed in gastric cancer cell lines AGS, BGC823, SGC-7901, MKN45, HGC27, MGC-803 and MKN28 at both mRNA and protein levels. The established AGS/CENP-H cell line with increased CENP-H expression showed enhanced proliferative activity, while the cell line MGC-803/CENP-H-RNAi with CENP-H expression interference showed an obviously lowered proliferation ability. CONCLUSION: CENP-H promotes the proliferation of human gastric cancer cells, suggesting its important role in the occurrence and development of gastric cancer.